Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. The Serine/threonine protein kinase ULK2 (unc-51 like kinase 2) plays an important regulatory role in autophagy thanks to its involvement in mTOR-regulated-initiation and downstream ATG protein-related progression of this catabolic process. An increasing number of miRNAs have been found to modulate autophagy by targeting some ATG genes. In this study, we focus on the role of mir-26b in autophagy in prostate cancer (PCa) cells. We found that miR-26b inhibited autophagy in PC-3 and C4-2 cells, through down-regulation of ULK2 expression. Dual luciferase reporter assays showed that miR-26b binds the 3'UTR of ULK2, suggesting that ULK2 is a direct target of miR-26b. Real-time PCR and Western blot analysis confirmed that over-expression of miR-26b reduced ULK2 mRNA and protein levels. Our results showed also that miR-26b was down-regulated in LNCaP, DU145, C4-2 and PC-3 cells compared to the two normal prostate cells RWPE-1 and WPMY-1 except DU145 cells. This inversely correlates with ULK2 level in the same cell lines. Expression level of ULK2 in tissues microarray (TMA) of prostate cancer derived from 96 patients positively correlated with the pathologic stage of the patients (*P < 0.05). Over-expression of ULK2 significantly reversed miR-26b-mediated autophagy inhibition. Taken together, our findings indicate that mir-26b inhibits autophagy through targeting ULK2 which is up-regulated in PCa. (C) 2016 The Authors. Published by Elsevier Inc.