To clarify response mechanisms to external Ser depletion in cells expressing Phgdh, we examined gene expression, intracellular Ser level, and cell proliferation in a mouse hepatoma Hepa1-6 line expressing Phgdh. Here, we demonstrate that extracellular Ser depletion causes transient transcriptional activation of Atf4-target genes Chac1, which associates with subsequent recovery of intracellular Ser levels and Gly consumption by upregulation of Phgdh and Shmt2. Here, SHMT2 is linked to hepatocellular carcinoma.