The Major Facilitator Superfamily (MFS) is a large and diverse group of transport proteins that facilitate the uptake and excretion of essential nutrients and ions, as well as the communication between cells and the environment. These proteins are crucial for energy-generating and energy-consuming processes in living organisms. The MFS includes both primary and secondary transporters, with primary transporters using ATP hydrolysis, photon absorption, electron flow, substrate decarboxylation, or methyl transfer to drive solute accumulation or extrusion. Secondary transporters, on the other hand, use chemiosmotic energy generated from primary transporters to facilitate the transport of additional solutes. Recent genome sequencing and biochemical studies have identified over 100 families of transport proteins, with the MFS and ATP-binding cassette (ABC) superfamily being the most prevalent. The MFS consists of single-polypeptide secondary carriers that transport small solutes in response to chemiosmotic ion gradients, while the ABC family includes multicomponent primary active transporters capable of transporting both small and macromolecules. The MFS was initially believed to primarily function in sugar uptake, but subsequent studies revealed its involvement in drug efflux systems, Krebs cycle metabolites, organophosphate/phosphate exchangers, and oligosaccharide/H+ symporters. The family has since expanded to include a wide range of functions and organisms, including bacteria, archaea, eukaryotic protists, fungi, animals, and plants. This report presents a comprehensive analysis of the MFS, constructing phylogenetic trees to classify its members into 17 distinct families based on sequence similarity. Each family is characterized by its specific substrate and function, with some families showing evidence of convergent evolution. The study also highlights the importance of the MFS in cell physiology and provides a rational classification system for these transport proteins. The findings underscore the significance of the MFS in the development of structural and functional diversity in living organisms.The Major Facilitator Superfamily (MFS) is a large and diverse group of transport proteins that facilitate the uptake and excretion of essential nutrients and ions, as well as the communication between cells and the environment. These proteins are crucial for energy-generating and energy-consuming processes in living organisms. The MFS includes both primary and secondary transporters, with primary transporters using ATP hydrolysis, photon absorption, electron flow, substrate decarboxylation, or methyl transfer to drive solute accumulation or extrusion. Secondary transporters, on the other hand, use chemiosmotic energy generated from primary transporters to facilitate the transport of additional solutes. Recent genome sequencing and biochemical studies have identified over 100 families of transport proteins, with the MFS and ATP-binding cassette (ABC) superfamily being the most prevalent. The MFS consists of single-polypeptide secondary carriers that transport small solutes in response to chemiosmotic ion gradients, while the ABC family includes multicomponent primary active transporters capable of transporting both small and macromolecules. The MFS was initially believed to primarily function in sugar uptake, but subsequent studies revealed its involvement in drug efflux systems, Krebs cycle metabolites, organophosphate/phosphate exchangers, and oligosaccharide/H+ symporters. The family has since expanded to include a wide range of functions and organisms, including bacteria, archaea, eukaryotic protists, fungi, animals, and plants. This report presents a comprehensive analysis of the MFS, constructing phylogenetic trees to classify its members into 17 distinct families based on sequence similarity. Each family is characterized by its specific substrate and function, with some families showing evidence of convergent evolution. The study also highlights the importance of the MFS in cell physiology and provides a rational classification system for these transport proteins. The findings underscore the significance of the MFS in the development of structural and functional diversity in living organisms.