Background: Osteoarthritis (OA) is a frequent and incurable joint disease, inducing significant pain and seriously threatening to human health. It has been reported that microRNAs (miRNAs) play crucial role on cancers and inflammatory diseases via cooperating with genes. However, the effect of miR-374a-3p/Wingless-type MMTV integration site family, member 5B (WNT5B) pair in OA remains to be explored. Methods: GSE105027 and GSE55457 were obtained to reveal the expression of miR-374a-3p and WNT5B in OA cartilages. The OA cell model was established by lipopolysaccharides (LPS) stimulation in CHON-001 cells and the functional role of miR-374a-3p on OA was investigated by analyzing cell proliferation, cell apoptosis and apoptosis-related proteins (Bcl-2, Bax and Bim) expression. Through bioinformatics prediction, WNT5B, the target gene of miR-374a-3p, was confirmed and the relationship between miR-374a-3p and WNT5B was further examined by luciferase reporter assay. Functional experiments in vitro were conducted to assess whether WNT5B was involved in the regulation of miR-374a-3p to LPS-stimulated CHON-001. Finally, JNK/ERK/MAPK pathway-related proteins expression was detected to explore the underlying molecular mechanism. Results: The data sets showed that miR-374a-3p was decreased in OA cartilages whilst WNT5B was increased. The consistent expressional pattern was observed in LPS-stimulated CHON-001 cells. Overexpression of miR-374a-3p significantly alleviated LPS-induced damage in CHON-001 cells. Further experiments demonstrated that WNT5B was a direct target of miR-374a-3p and its expression was decreased by miR-374a-3p. Down-regulation of WNT5B strengthened the protective effect of miR-374a-3p on LPS-stimulated CHON-001 cells. Moreover, miR-374a-3p impaired the JNK/ERK/MAPK pathway by regulating WNT5B in OA cell model. Conclusion: These results indicated that overexpression of miR-374a-3p protects CHON-001 cells against LPS challenge by inhibiting the JNK/ERK/MAPK pathway through modulating WNT5B.