Metastasis is the main cause of cancer-specific death in patients with prostate cancer (PCa). Acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) is involved in the metabolic reprogramming of multiple types of cancer cells, but its role in PCa metastasis remains largely unknown. Here, we determined the effect of overexpression or small interfering RNA-mediated depletion of ACSL3 on the migratory and invasive abilities of human PCa cell lines. We also conducted phospho-protein microarray analysis to identify signaling pathway components affected by ACSL3 modulation. Overexpression of ACSL3 promoted the migration and invasion of PCa cells, whereas ACSL3 downregulation had the opposite effects. Mechanistically, phospho-protein analysis showed that ACSL3 regulated the phosphorylation of AKT and the expression of matrix metalloproteinase9. Our results support a potential role for ACSL3 in promoting the metastatic behavior of PCa, possibly via AKT/matrix metalloproteinase9 pathways. Thus, ACSL3 could be a novel target for the development of treatments for PCa.