Persistent inflammasome activation contributes to chronic, low grade inflammation. However, it is unclear how the inflammasome activation is sustained after initiation. Here we reported that N4-acetylcytidine (N4A), a nucleoside metabolite, activated microglia and sustained NLRP3 inflammasome activation by inducing HMGB1 signaling. Released HMGB1 through N4A activated NFκB and induced NLRP3 expression. HMGB1 silencing abolished N4A-stimulated NFκB activation, NLRP3 and persistent HMGB1 expression. In addition, inhibiting NLRP3 expression by RNAi abrogated N4A-mediated HMGB1 expression. Lack of NLRP3 inflammasome adaptor named apoptosis-associated speck-like protein containing a CARD (ASC) abrogated N4A-induced HMGB1 expression, NFκB activation, and NLRP3 expression. Taken together, our results reveal a novel role of N4A in activation of NLRP3 inflamasome via HMGB1 feedback.