Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. In *Salmonella*, central metabolic enzymes are extensively acetylated in response to different carbon sources, with acetylation levels changing in concert with cell growth and metabolic flux. Key enzymes controlling glycolysis versus gluconeogenesis and the citrate cycle versus glyoxylate bypass are regulated by acetylation. This regulation is mainly controlled by a pair of lysine acetyltransferase (Pat) and deacetylase (CobB), whose expressions are coordinated with growth status. Reversible acetylation of metabolic enzymes allows cells to respond to environmental changes by sensing cellular energy status and adjusting reaction rates or directions. This represents a conserved metabolic regulatory mechanism from bacteria to mammals. Protein lysine acetylation regulates a wide range of cellular functions in eukaryotes, including transcriptional control. In prokaryotes like *Salmonella*, reversible lysine acetylation regulates the activity of acetyl-CoA synthetase. To determine how lysine acetylation globally regulates metabolism in prokaryotes, researchers analyzed the acetylation status of *Salmonella* proteins under different carbon sources. They identified 235 acetylated peptides corresponding to 191 proteins, with about 50% of acetylated proteins involved in multiple metabolic pathways and 90% of central metabolism enzymes acetylated. SILAC analysis showed that enzymes with altered acetylation in response to different carbon sources showed greater acetylation in glucose-grown cells than in citrate-grown cells. Acetylation of central metabolic enzymes in a *pat* null mutant was reduced, while in a *cobB* null mutant, acetylation was elevated. Acetylation of ribosomal proteins was unchanged in both mutants, supporting the notion that Pat and CobB are the major enzymes responsible for reversible acetylation of central metabolic enzymes in *Salmonella*. The physiological role of enzyme acetylation in mediating cellular adaptation to different metabolic fuels was suggested by the different growth properties of wild-type, *Δpat*, or *ΔcobB* strains grown on different carbon sources. The regulatory role of Pat and CobB in coordinating carbon source adaptation was tested by measuring in vivo metabolic flux profiles with 13C-labeled glucose or citrate as tracers. The overall metabolic flux profiles in *Salmonella* showed distinct patterns during growth on glucose or citrate. The glyoxylate bypass was activated, and carbon flow favored gluconeogenesis when cells were grown on citrate. Quantitative comparison of flux profiles through glycolysis, gluconeogenesis, TCA cycle, and glyoxylate bypass of wild-type, *Δpat*, and *ΔcobB* strains showed that acetylation status affects metabolic flux profiles. Direct evidence of acetylation-mediated regulation for central metabolic enzymes was derived from biochemical studiesAcetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. In *Salmonella*, central metabolic enzymes are extensively acetylated in response to different carbon sources, with acetylation levels changing in concert with cell growth and metabolic flux. Key enzymes controlling glycolysis versus gluconeogenesis and the citrate cycle versus glyoxylate bypass are regulated by acetylation. This regulation is mainly controlled by a pair of lysine acetyltransferase (Pat) and deacetylase (CobB), whose expressions are coordinated with growth status. Reversible acetylation of metabolic enzymes allows cells to respond to environmental changes by sensing cellular energy status and adjusting reaction rates or directions. This represents a conserved metabolic regulatory mechanism from bacteria to mammals. Protein lysine acetylation regulates a wide range of cellular functions in eukaryotes, including transcriptional control. In prokaryotes like *Salmonella*, reversible lysine acetylation regulates the activity of acetyl-CoA synthetase. To determine how lysine acetylation globally regulates metabolism in prokaryotes, researchers analyzed the acetylation status of *Salmonella* proteins under different carbon sources. They identified 235 acetylated peptides corresponding to 191 proteins, with about 50% of acetylated proteins involved in multiple metabolic pathways and 90% of central metabolism enzymes acetylated. SILAC analysis showed that enzymes with altered acetylation in response to different carbon sources showed greater acetylation in glucose-grown cells than in citrate-grown cells. Acetylation of central metabolic enzymes in a *pat* null mutant was reduced, while in a *cobB* null mutant, acetylation was elevated. Acetylation of ribosomal proteins was unchanged in both mutants, supporting the notion that Pat and CobB are the major enzymes responsible for reversible acetylation of central metabolic enzymes in *Salmonella*. The physiological role of enzyme acetylation in mediating cellular adaptation to different metabolic fuels was suggested by the different growth properties of wild-type, *Δpat*, or *ΔcobB* strains grown on different carbon sources. The regulatory role of Pat and CobB in coordinating carbon source adaptation was tested by measuring in vivo metabolic flux profiles with 13C-labeled glucose or citrate as tracers. The overall metabolic flux profiles in *Salmonella* showed distinct patterns during growth on glucose or citrate. The glyoxylate bypass was activated, and carbon flow favored gluconeogenesis when cells were grown on citrate. Quantitative comparison of flux profiles through glycolysis, gluconeogenesis, TCA cycle, and glyoxylate bypass of wild-type, *Δpat*, and *ΔcobB* strains showed that acetylation status affects metabolic flux profiles. Direct evidence of acetylation-mediated regulation for central metabolic enzymes was derived from biochemical studies