Musashi-1 (MSI1) has been proposed as a potential prognostic biomarker in prostate cancer (PCa), but its role and underlying molecular mechanisms in PCa progression remain unclear. The mRNA and protein levels of MSI1 and α/β-hydrolase domain 2 (ABHD2) in PCa tissues and cells were examined using qRT-PCR and western blot. Cell proliferation, cycle, apoptosis, and migration were detected by EdU assay, flow cytometry and transwell assay. Glucose uptake and lactate production were assessed to measure cell glycolysis. The interaction between SP1 and PLA2G6 was evaluated using dual-luciferase reporter assay and ChIP assay. MSI1 had increased expression in PCa tissues and cells. MSI1 downregulation could repress PCa cell proliferation, cycle, migration, glycolysis, and enhanced apoptosis. ABHD2 was upregulated in PCa tissues and cells, and MSI1 could bind to ABHD2 promoter region to increase its expression. Knockdown of ABHD2 suppressed PCa cell growth, migration and glycolysis, and ABHD2 overexpression also abolished the effect of MSI1 downregulation on PCa cell progression. Furthermore, interference of MSI1 reduced PCa tumor growth by decreasing ABHD2 expression in vivo. MSI1 facilitated PCa cell proliferation, migration and glycolysis via activating ABHD2 transcription, providing a novel target for PCa treatment.