<p>A large-scale longitudinal epidemiological study by the Hisayama study revealed that the concentration of one-carbon metabolism-related amino acids in the serum changes with age and that there is a link between these fluctuations and the risk of developing dementia (Hata et al. in Am J Epidemiol 188:1637–1645, 2019; Mihara et al. in Sci Rep 12:12427, 2022). Therefore, the aim of this study was to focus on age-related changes in one-carbon metabolism-related amino acids and elucidate the regulatory basis of these changes. Treatment with etoposide, an anti-cancer drug, induced cellular senescence in SH-SY5Y cells, as indicated by increased senescence-associated β galactosidase activity and upregulated expression of senescence markers <i>p16</i> and <i>p21</i>. Liquid chromatography-mass spectrometry analysis revealed that the intracellular amino acid concentrations, particularly those involved in the one-carbon metabolism, were elevated in senescent cells, including those of methionine, S-adenosylmethionine, S-adenosylhomocysteine (SAH), homocysteine (Hcys), and related metabolites. The results of the expression analysis focused on the enzyme genes involved in Hcys metabolism and revealed that the induction of cellular senescence upregulated <i>adenosylhomocysteinase like 1/2</i> (<i>AHCYL1/L2</i>), which convert SAH to Hcys. Additionally, the genes involved in Hcys metabolism via the sulphuration pathway (<i>KYAT1/3</i> and <i>CTH</i>) were significantly upregulated. Because Hcys has been implicated in aging, further investigations focused on <i>AHCYL1/L2</i>. Gene knockdown of <i>AHCYL1/L2</i> in etoposide-treated cells reduced <i>p16</i> and <i>p21</i> expression, indicating that <i>AHCYL1/L2</i> is essential for cellular senescence induction. These findings suggest that Hcys accumulation and its metabolic enzymes play a crucial role in cellular senescence.</p>