High-resolution atomic force microscopy (AFM) was used for the in situ evaluation of the anti-inflammatory effects of triptolide on rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to understand the anti-RA effects of triptolide, based on the morphological and biophysical changes observed in RA-FLS. RA-FLS have been reported to play a primary role in inflammatory bone destruction during the development of RA and thus are regarded as an important target for RA treatment. Triptolide pretreatment significantly inhibited tumor necrosis factor-α-induced expression of the interleukin (IL)-1β, IL-6, and IL-8 genes in MH7A cells. Using AFM, we showed that triptolide-induced morphological damage in MH7A cells by inducing significant ultrastructure changes in the membrane, which were closely related to triptolide-induced apoptosis in MH7A cells. Using force measurements determined with AFM, triptolide was shown to increase the stiffness of MH7A cells. These findings not only revealed the strong anti-inflammatory effects of triptolide on RA-FLS, highlighting triptolide as a potential anti-RA agent, but also revealed the possible use of AFM for studying anti-inflammatory responses in RA-FLS, which we expect to be developed into a potential tool for anti-RA drug studies in RA-FLS.