Abstract Body: Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death worldwide, despite effective lipid-lowering drugs. There is an urgent need to identify alternative metabolic pathways to target for ASCVD treatment. Recent studies establish dysregulated amino acid metabolism in ASCVD. Notably, glycine is the only amino acid consistently found at significantly lower circulating levels in patients with ASCVD, and glycine-based treatment lowers atherosclerosis in mice. Recently, we reported that glycine depletion is driven by hepatocyte serine hydroxymethyltransferase 2 (SHMT2), which catalyzes the interconversion of glycine and serine. Importantly, a lower glycine:serine ratio correlates with ASCVD severity in humans. Yet, the role of hepatic SHMT2 in ASCVD has not been systematically investigated. To address this gap of knowledge, we generated atherogenic mice lacking hepatocyte Shmt2 (Shmt2^HKO/Apoe^-/-) and fed them a Western diet for 12 weeks. Endpoint analyses were based on metabolomics, lipidomics, stable-isotope tracing, and transcriptomics, combined with biochemical and histological studies. Metabolomics revealed a significant increase in glycine and the glycine:serine ratio in plasma from Shmt2^HKO/Apoe^-/- mice compared to Shmt2^flox/Apoe^-/- littermates. In livers from Shmt2^HKO/Apoe^-/- mice, decreased S-adenosylmethionine (SAM) and increased adenosylhomocysteine (SAH) resulted in reduced SAM:SAH ratio. While hepatic steatosis was unaltered, untargeted lipidomics highlighted alternations in phospholipids involved in lipoprotein assembly. Specifically, Shmt2^HKO/Apoe^-/- mice had lower hepatic phosphatidylcholine (PC) and (PC):phosphatidylethanolamine (PE) ratio, key regulators of VLDL assembly and secretion. Accordingly, plasma total cholesterol and VLDL-cholesterol were significantly decreased in Shmt2^HKO/Apoe^-/- mice, independent of sex. The lipidomics further highlighted a decrease in hepatic ceramides in Shmt2^HKO/Apoe^-/- mice. In line, stable-isotope tracing studies uncovered a significant decrease in de novo synthesis of sphinganine, the rate-limiting ceramide precursor. Furthermore, unbiased RNA-Seq, validated by qRT-PCR, confirmed a suppression of proinflammatory mediators (Tnfα, Ir1b, Ccr5). Finally, histological analysis of aortic sinuses revealed lower plaque size in Shmt2^HKO/Apoe^-/- mice. Altogether, our studies uncover hepatic SHMT2 as a driver of atherosclerosis through one-carbon metabolic regulation of VLDL and ceramides.