Fatty acid oxidation supports natural killer (NK) cell responses against infection and cancer. NK cells, crucial for innate immunity, rely on fatty acid oxidation (FAO) for metabolic resilience and function. This study shows that activated NK cells, upon exposure to viral infection or cancer, increase fatty acid uptake and express carnitine palmitoyltransferase I (CPT1A), a key enzyme for long-chain fatty acid oxidation. CPT1A-deficient NK cells exhibit reduced mitochondrial function, impaired actin cytoskeleton rearrangement, and diminished antitumor and antiviral activity. Using a mouse model with NK cell-specific CPT1A deletion and stable isotope tracing, the study demonstrates that FAO is essential for maintaining mitochondrial homeostasis, supporting the TCA cycle, and producing aspartate, which is critical for DNA, RNA, and protein synthesis. CPT1A is vital for NK cell proliferation during viral infection, but not for CD8+ T cells. CPT1A-deficient NK cells show reduced cytotoxicity and protection against cancer, highlighting the importance of FAO in NK cell function. The study also reveals that FAO supports actin rearrangement, which is essential for NK cell cytotoxicity against tumor targets. These findings underscore the role of FAO in NK cell metabolism and suggest that optimizing FAO could enhance NK cell-based immunotherapies for cancer and viral infections.Fatty acid oxidation supports natural killer (NK) cell responses against infection and cancer. NK cells, crucial for innate immunity, rely on fatty acid oxidation (FAO) for metabolic resilience and function. This study shows that activated NK cells, upon exposure to viral infection or cancer, increase fatty acid uptake and express carnitine palmitoyltransferase I (CPT1A), a key enzyme for long-chain fatty acid oxidation. CPT1A-deficient NK cells exhibit reduced mitochondrial function, impaired actin cytoskeleton rearrangement, and diminished antitumor and antiviral activity. Using a mouse model with NK cell-specific CPT1A deletion and stable isotope tracing, the study demonstrates that FAO is essential for maintaining mitochondrial homeostasis, supporting the TCA cycle, and producing aspartate, which is critical for DNA, RNA, and protein synthesis. CPT1A is vital for NK cell proliferation during viral infection, but not for CD8+ T cells. CPT1A-deficient NK cells show reduced cytotoxicity and protection against cancer, highlighting the importance of FAO in NK cell function. The study also reveals that FAO supports actin rearrangement, which is essential for NK cell cytotoxicity against tumor targets. These findings underscore the role of FAO in NK cell metabolism and suggest that optimizing FAO could enhance NK cell-based immunotherapies for cancer and viral infections.