Non-small cell lung cancer (NSCLC) is a subtype of the most frequently diagnosed cancer, causing a considerable number of deaths globally. Mitochondrial dysfunction was found to promote malignant progression. However, the underlying mechanism remains unclear. Acyl-CoA synthetase short chain family member 3 (ACSS3) is mainly located in mitochondria, which abnormal regulation is usually accompanied by the occurrence and development of tumors. In this study, we found that the expression level of ACSS3 was correlated with poor prognosis in patients with NSCLC. Moreover, we demonstrated that ACSS3 knockdown led to mitochondrial contraction, increased reactive oxygen species levels, decreased mitochondrial membrane potential, and subsequently inhibited tumor growth of NSCLC cells in vitro and in vivo, whereas its overexpression promoted these processes. Mechanistically, ACSS3 knockdown promoted ferroptosis through transcriptional control of SLC7A11 and GPX4. Further investigations indicated that ACSS3 loss inhibited the SLC7A11/GPX4 axis by enhancing p53 stability. Taken together, our data confirmed that ACSS3 promotes NSCLC tumorigenesis through inhibiting the p53-mediated ferroptosis. Hence, ACSS3 emerges as a promising therapeutic target for NSCLC treatment.