Increasing evidence suggests an important role for amino acid metabolism in atherosclerotic cardiovascular disease (ASCVD), highlighting potential biomarkers, causative factors, and therapeutic targets. Among all amino acids, lower circulating glycine is consistently reported to be associated with enhanced ASCVD severity. The amino acid serine is the main precursor for glycine, and a lower glycine:serine ratio is associated with more advanced and unstable atherosclerotic plaques. The mitochondrial serine hydroxymethyltransferase 2 (SHMT2) catalyzes the bulk of glycine formation from serine, and is critical in development since global knockout mice are nonviable. We found that SHMT2 is highly expressed in macrophages in both human and mouse atherosclerotic plaques. LPS-treated macrophages displayed reduced SHMT2 expression concomitant with a lower intracellular glycine:serine ratio. To study the role of SHMT2 in macrophages, we developed novel myeloid-specific Shmt2 knockout mice ( Shmt2 MKO ). Metabolomics and stable isotope tracing studies revealed that macrophages lacking SHMT2 have a lower glycine:serine ratio leading to attenuated de novo synthesis of glutathione and enhanced formation of the ceramide precursor, sphinganine. Accordingly, the loss of SHMT2 in macrophages enhanced superoxide production and ceramide accumulation. To determine the role of macrophage SHMT2 in atherosclerosis, we crossed the Shmt2 MKO mice with apolipoprotein E-deficient ( Apoe -/- ) mice. After 12 weeks on Western diet, Shmt2 MKO / Apoe -/- mice showed no differences in plasma lipid or amino acid profiles as compared to Shmt2 flox / Apoe -/- mice. Nevertheless, plaque area and necrotic core size were significantly enhanced in Shmt2 MKO / Apoe -/- mice. Taken together, macrophage SHMT2 plays a critical role in atherosclerosis by regulating redox and ceramide metabolism.