l ‐Serine ( l ‐Ser) is a necessary precursor for the synthesis of proteins, lipids, glycine, cysteine, d ‐serine, and tetrahydrofolate metabolites. Low l ‐Ser availability activates stress responses and cell death; however, the underlying molecular mechanisms remain unclear. l ‐Ser is synthesized de novo from 3‐phosphoglycerate with 3‐phosphoglycerate dehydrogenase (Phgdh) catalyzing the first reaction step. Here, we show that l ‐Ser depletion raises intracellular H 2 O 2 levels and enhances vulnerability to oxidative stress in Phgdh‐deficient mouse embryonic fibroblasts. These changes were associated with reduced total glutathione levels. Moreover, levels of the inflammatory markers thioredoxin‐interacting protein and prostaglandin‐endoperoxide synthase 2 were upregulated under l ‐Ser‐depleted conditions; this was suppressed by the addition of N ‐acetyl‐ l ‐cysteine. Thus, intracellular l ‐Ser deficiency triggers an inflammatory response via increased oxidative stress, and de novo l ‐Ser synthesis suppresses oxidative stress damage and inflammation when the external l ‐Ser supply is restricted.