ATP citrate lyase (ACLY) is a key enzyme that is involved in de novo lipogenesis by catalyzing conversion of cytosolic citrate into acetyl CoA and oxaloacetate. Up-regulation of ACLY in various types of tumors enhances fatty acid synthesis and supplies excess acetyl CoA for histone acetylation. However, there is evidence that its enzymatic activity alone is insufficient to explain ACLY silencing-mediated growth arrest in tumor cells. In this study, we found that ACLY knockdown in primary human cells triggers cellular senescence and activation of tumor suppressor p53. Provision of acetyl CoA to ACLY knockdown cells did not alleviate ACLY silencing-induced p53 activation, suggesting an independent role for ACLY activity. Instead, ACLY physically interacted with the catalytic subunit of AMP-activated protein kinase (AMPK) and inhibited AMPK activity. The activation of AMPK under ACLY knockdown conditions may lead to p53 activation, ultimately leading to cellular senescence. In cancer cells, ACLY silencing-induced p53 activation facilitated DNA damage-induced cell death. Taken together, our results suggest a novel function of ACLY in cellular senescence and tumorigenesis.