Increasing evidence supports the role of microRNAs (miRNA) in the regulation of inflammation in various human disorders. Several recent studies have demonstrated that microRNA-25 (miR-25) has multiple functions, and it affects the expression of inflammatory mediators. Withaferin A (WFA), a natural compound derived from the medicinal plant Withania somnifera, has shown the potential to be an effective drug for arthritis treatment in several preclinical and clinical studies. We investigated the role of miR-25 in the WFA-mediated up-regulation of cyclooxygenase-2 (COX-2) expression in rabbit articular chondrocytes. WFA induced COX-2 expression in a dose-dependent manner as analyzed by western blot analysis and immunofluorescence staining in rabbit articular chondrocytes. WFA up-regulated miR-25 expression as determined by real-time PCR. Overexpression of miR-25 in the presence of WFA increased the expression of COX-2 compared to that observed with just WFA treatment alone, as indicated by western blot analysis and Real-time PCR. Moreover, silencing of miR-25 by anti-miR25 inhibited COX-2 expression in a dose-dependent manner. Since miR-25 up-regulation by WFA treatment induced the expression of COX-2 in rabbit articular chondrocytes, these findings collectively suggest that miR-25 mediates the WFA-induced inflammatory responses in chondrocytes.