Senescence, a cellular process through which damaged or dysfunctional cells suppress the cell cycle, contributes to aging or age-related functional decline. Cell metabolism has been closely correlated with aging processes, and it has been widely recognized that metabolic changes underlie the cellular alterations that occur with aging. Here, we report that fatty acid oxidation (FAO) serves as a critical regulator of cellular senescence and uncover the underlying mechanism by which FAO inhibition induces senescence. Pharmacological or genetic ablation of FAO results in a p53-dependent induction of cellular senescence in human fibroblasts, whereas enhancing FAO suppresses replicative senescence. We found that FAO inhibition promotes cellular senescence through acetyl-CoA, independent of energy depletion. Mechanistically, increased formation of autophagosomes following FAO inhibition leads to a reduction in SIRT1 protein levels, thereby contributing to senescence induction. Finally, we found that inhibition of autophagy or enforced expression of SIRT1 can rescue the induction of senescence as a result of FAO inhibition. Collectively, our study reveals a distinctive role for the FAO-autophagy-SIRT1 axis in the regulation of cellular senescence. [BMB Reports 2023; 56(12): 651-656].