WIN-34B showed analgesic and anti-inflammatory effects in various animal models of pain and osteoarthritis. However, the molecular mechanism by which WIN-34B inhibits pain and inflammation in vivo remains to be elucidated. We investigated the molecular mechanisms of the actions of WIN-34B using various in vitro models using fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA FLSs), RAW264.7 cells and peritoneal macrophages. WIN-34B inhibited the level of IL-6, $PGE_2$, and MMP-13 in IL-$1{eta}$-stimulated RA FLSs in a dose-dependent manner. The mRNA levels were also inhibited by WIN-34B. The level of $PGE_2$, NO, IL-$1{eta}$, and TNF-${alpha}$ were inhibited by WIN-34B at different concentrations in LPS-stimulated RAW264.7 cells. The production of NO and $PGE_2$ was inhibited by WIN-34B in a dose-dependent manner in LPS-stimulated peritoneal macrophages. All of these effects were comparable to the positive control, celecoxib or indomethacin. I${kappa}B$B signaling pathways were inhibited by WIN-34B, and the migration of NF-${kappa}B$ into the nucleus was inhibited, which is consistent with the degradation of $I{kappa}B-{alpha}$. Taken together, the results suggest that WIN-34B has potential as a therapeutic drug to reduce pain and inflammation by inhibiting the production of pro-inflammatory mediators.