Osteoarthritis (OA) is a complex, degenerative, multi-factorial joint disease. Because of the difficulty in treating OA, developing new targeting strategies that can be used to understand its molecular mechanisms is critical. Protaetia brevitarsis seulensis larvae offer much therapeutic value; however, the presence of various active compounds and the multi-factorial risk factors for OA render the precise mechanisms of action unclear. A systematic transcriptome analysis was used to investigate the key mechanisms of action of P. brevitarsis seulensis larvae aqueous extract (PBSL) and its compounds on OA. Major mechanisms and transcription factors of PBSL were analyzed by profiling gene expression changes in interleukin (IL)-1β-induced human chondrosarcoma cell (SW1353) treated with PBSL. An in vitro assay was performed to validate the efficacy of the novel mechanism and targets of PBSL. PBSL exerted anti-inflammatory effects on SW1353 cells by regulating many molecular pathways. The IL-6/JAK/STAT3 pathway was significantly downregulated by PBSL, and STAT3 was identified as a major transcription factor regulating PBSL-induced target gene expression. Of the six PBSL compounds, the major compound was regulated by the IL-6/JAK/STAT3 pathway. This study provided potential novel mechanisms and transcription factors for PBSL and its active compounds against OA and indicated that inhibiting the IL-6/JAK/STAT3 pathway is a therapeutic target for treating OA.