Objective To investigate the role and underlying mechanism of miR-15b-5p on hypoxia/reoxygenation (H/R) induced human renal tubular epithelial cell (HK-2) injury by targeting forkhead box O1 (FOXO1). Methods HK-2 cells in the log growth phase were set up as follows: (1) control group (normal culture) and H/R group (H/R induced culture). The expressions of miR-15b-5p and FOXO1 mRNA were detected using qRT-PCR, and the protein expression of FOXO1 was detected using Western blotting. (2) Control group (normal culture), H/R group (H/R induced culture), H/R+mimic control group (cells transfected with mimic control then induced by H/R), H/R+miR-15b-5p mimic group (cells transfected with miR-15b-5p mimic then induced by H/R), H/R+miR-15b-5p mimic+OE-NC group (cells co-transfected with miR-15b-5p mimic and OE-NC plasmid, then induced by H/R), and H/R+miR-15b-5p mimic+OE-FOXO1 group (cells co-transfected with miR-15b-5p mimic and FOXO1 overexpression plasmid, then induced by H/R). The expression of miR-15b-5p was detected using qRT-PCR, and the protein expressions of FOXO1, cleaved caspase-3, Bax, and Bcl-2 were detected using Western blotting. CCK-8 assay was used to detect cell viability. Cell apoptosis was measured by the TUNEL method. (3) Control group (normal culture), H/R group (H/R induced culture), H/R+miR-15b-5p mimic group (cells transfected with miR-15b-5p mimic then induced by H/R), and H/R+miR-15b-5p mimic+OE-FOXO1 group (cells co-transfected with miR-15b-5p mimic and FOXO1 overexpression plasmid, then induced by H/R). The protein expressions of LC3, p62 and Beclin1 were detected using Western blotting. LC3 immunofluorescence was used to detect the cell autophagy. The target reaction between miR-15b-5p and FOXO1 was assessed using dual luciferase reporting assay. Results Under an inverted microscope, it was observed that the control group had a higher number of cells, most of which were in a typical cobblestone shape and grew in a cobblestone-like manner; most of the cells in the H/R group contracted and became round, with a significant decrease in the number of adherent cells. In H/R-induced HK-2 cells, miR-15b-5p was significantly down-regulated, while miRNA and protein expression of FOXO1 was up-regulated (P<0.05). Luciferase assay results showed that miR-15b-5p directly targeted the 3'-UTR of FOXO1. Overexpression of miR-15b-5p increased cell viability, reduced cell apoptosis, and decreased autophagy in H/R-induced HK-2 cells (P<0.05). Compared with H/R+miR-15b-5p mimic group, the viability of HK-2 cells was decreased, the apoptosis and autophagy level were increased in H/R+miR-15b-5p mimic+OE-FOXO1 group (P<0.05). Conclusion miR-15b-5p inhibited autophagy and alleviated H/R-induced HK-2 cell injury by targeting FOXO1.