The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) contributes to cartilage and bone destruction in rheumatoid arthritis (RA). Betulinic acid has been demonstrated to have a positive therapeutic effect on tumor, inflammation and immune disorder, however, the effects of betulinic acid on RA FLSs have not been verified. Therefore, in the present study, we observed the effect of betulinic acid on the migration and invasion of RA FLSs and explored its underlying signal mechanisms. Our results showed that betulinic acid treatment suppressed the migration, invasion and reorganization of the actin cytoskeleton of RA FLSs. In addition, we found that the mRNA expression of IL-1β, IL-6, IL-8 and IL-17A were markedly down-regulated by treatment with betulinic acid in TNF-α-induced RA FLSs. To gain insight into the molecular mechanisms, we evaluated the effect of betulinic acid on NF-κB activation in RA FLSs. The results indicated that betulinic acid treatment reduced the TNF-α-induced activation of NF-κB signal pathway and the NF-κB nuclear accumulation. We also observed that treatment with betulinic acid attenuated synovial inflammation and joint destruction in mice with CIA. Taken together, these results suggest that betulinic acid inhibits the migration and invasion of RA FLSs by blocking NF-κB signal pathway activation.