In this study, we investigated whether the anti-inflammatory effects of tomatidine alleviate osteoarthritis (OA)-related pathology in primary articular chondrocytes and a rat OA model. STITCH database analysis identified 22 tomatidine-target genes that were enriched in 78 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Moreover,39 of the 105 OA-related KEGG pathways were related to tomatidine-target genes. The top two OA-related KEGG pathways with tomatidine-target genes were the MAPK and neutrophin signaling pathways. Pretreating primary chondrocytes with tomatidine suppressed interleukin-1β (IL-1β)-induced expression of iNOS, COX-2, MMP1, MMP3, MMP13, and ADAMTS-5. Tomatidine also suppressed IL-1β-induced degradation of collagen-II and aggrecan proteins by inhibiting NF-κB and MAPK signaling. In a rat OA model, histological and immunohistochemical analyses showed significantly less cartilage degeneration in thetibiofemoral joints of rats treated for 12 weeks with tomatidine after OA induction (experimental group) than in untreated OA group rats. However, micro-computed tomography (μ-CT) showed that tomatidine did not affect remodeling of the subchondral bone at the tibial plateau. These data shows that tomatidine suppresses IL-1β-induced inflammation in primary chondrocytes by inhibiting the NF-κB and MAPK signaling pathways, and protects against cartilage destruction in a rat OA model.