The expression of hsa-miR-125b is significantly downregulated in the fluorouracil-resistant cell line of human gastric cancer (BGC823/Fu). In order to investigate the role of hsa-miR-125b in the drug-resistance acquisition process of human gastric cancer, we performed a series of analysis on the sequence characteristics, species conservation, target genes, function annotation and signal transduction pathway enrichment of hsa-miR-125b using a combined bioinformatic approach such as miRbase, TargetScan6.2, PicTar, miRanda, Gene Ontology(GO) and KEGG. The results showed that the sequence of miR-125b is highly conserved in multiple species. A total of 79 target genes related to transcription regulation, protein binding, enzyme activity (P<0.001) were predicted by bioinformatics software. These genes involved in many biological processes including cell cycle, cell proliferation, cell apoptosis and cell responses to cytokine, drug responses and DNA damage (P<0.001). And these target genes mainly belong to MAPK, Wnt and p53 signal transduction pathways (P<0.01). The results revealed that hsa-miR-125b may regulate multiple biological processes and signal transduction pathways, and drug-resistant occurrence is associated with cell proliferation, cell apoptosis, cell cycle and signaling pathways including MAPK, Wnt and p53. We suggest that hsa-miR-125b may affect chemosensitivity by regulating target genes involved in the above processes and these target genes might be reliable candidates for exploring the role of hsa-miR-125b in tumor chemoresistance.