The relationship between abnormal lipid acid metabolism and the progression of lung cancer is increasingly evident. Carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme in fatty acid oxidation, has been implicated in the advancement of various cancers. However, the role of CPT1A in lung cancer and the regulatory mechanisms of microRNAs on CPT1A-mediated fatty acid oxidation remain largely unknown. In our study, we demonstrate that miR-365-3p inhibits CPT1A expression by targeting its 3'-untranslated region in lung cancer cells. The inhibition of CPT1A by miR-365-3p leads to increased lipid droplet accumulation, diminished ATP production, and a decrease in fatty acid oxidation levels. Furthermore, the disruption of fatty acid oxidation attenuates the ability of the miR-365-3p/CPT1A axis to modulate lung cancer cell proliferation and migration both in vitro and in vivo. Clinical data reveal that CPT1A expression is significantly upregulated while miR-365-3p is markedly downregulated. Additionally, there exists a negative correlation between miR-365-3p and CPT1A expression, and both are predictive of clinical outcome in lung cancer patients. Collectively, our findings shed light on the function and mechanistic pathway of the miR-365-3p/CPT1A axis in lung cancer, which might provide a potential therapeutic target for lung cancer.