Increasing studies indicated that Cucurbitacin E (CuE), a compound isolated from Cucurbitaceae, has been shown anti-inflammatory effect. However, the effect of CuE on rheumatoid arthritis (RA) inflammatory response and its potential molecular mechanism are still unknown. In this study, we demonstrated that CuE significantly suppressed TNF-α-induced inflammatory cytokines production interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) mRNA and protein expression in human synoviocyte MH7A cells. Furthermore, we found that CuE also inhibited TNF-α-induced phosphorylation of NF-κBp65, IKKα/β, and IκBα in a dose-and time-dependent manner as well as NF-κBp65 nuclear translocation. Finally, we showed that CuE blocked the upstream targets of NF-κB pathway RIP1/PI3K/Akt. Interestingly, PI3K inhibitor LY294002 completely blocked the TNF-α-induced activation of p85, Akt and the whole cascade of the NF-κB signaling components and suppressed inflammatory cytokines production in mRNA and protein levels similarly as CuE. Our studies provided the first evidence that CuE inhibited TNF-α-induced inflammatory cytokine production in human synoviocyte MH7A cells via modulation of PI3K/Akt/NF-κB pathway. These findings indicated that CuE is a potential candidate for RA therapy.