Abstract Body: Atherosclerotic cardiovascular disease (ASCVD), a leading cause of death worldwide, is characterized by plaque buildup in arteries. While stable plaques are clinically silent, unstable plaques with large necrotic cores and thin fibrous caps can rupture, leading to heart attacks or strokes. The development of unstable plaques is driven by impaired macrophage clearance of apoptotic cells, contributing to necrotic core formation. Despite advances in lipid-lowering therapies, residual ASCVD risk persists, underscoring the need for alternative therapeutic targets. Emerging research highlights dysregulated amino acid metabolism, particularly glycine and serine, in ASCVD, yet the role of this metabolic pathway in macrophages and atherosclerotic plaque stability remains unexplored. The enzyme serine hydroxymethyltransferase-2 (SHMT2) catalyzes the conversion of serine to glycine. Glycine is a precursor to glutathione (GSH), the major intracellular antioxidant, while serine, combined with palmitoyl CoA, forms sphinganine, a precursor to ceramides. Both reactive oxygen species (ROS) and ceramides impair efferocytosis, driving necrotic core formation. We found that SHMT2 expression in macrophages decreases with atherosclerotic plaque severity in humans and in proinflammatory macrophages, aligned with reduced glycine:serine ratios. To study the role of SHMT2 in macrophages, we developed novel myeloid-specific Shmt2 knockout mice (Shmt2^MKO) using CRISPR/Cas9 and Cre-lox recombination. RNA sequencing revealed upregulation of sphingolipid metabolism and ROS pathways, with downregulation of phagosome-related pathways in SHMT2 deficient macrophages. Metabolomics and stable isotope tracing studies showed that loss of SHMT2 in macrophages inhibits de novo GSH synthesis and induces sphinganine formation, increases ROS and ceramides, and impairs efferocytosis. Interestingly, restoration of SHMT2 via stable mRNA reduces ROS and rescues defective efferocytosis. 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. Strikingly, larger necrotic cores and thinner fibrous caps were found in Shmt2^MKO / Apoe^-/- mice. Taken together, macrophage SHMT2 plays a critical role in atherosclerosis by regulating redox and ceramide metabolism.