The ATPase activity of NLRP3 has pivotal role in inflammasome activation and is recognized as a good target for the development of the NLRP3 inflammasome-specific inhibitor. However, signals in the vicinity of the ATPase activity of NLRP3 have not been fully elucidated. Here, we demonstrate NLRP3 inflammasome-specific action of a benzoxathiole derivative, BOT-4-one. BOT-4-one exhibited an inhibition of NLRP3 inflammasome activation, which was attributable to its alkylating capability to NLRP3. In particular, the NLRP3 alkylation by BOT-4-one led to an impaired ATPase activity of NLRP3, thereby obstructing the assembly of the NLRP3 inflammasome. Additionally, we found that NLRP3 alkylators, including BOT-4-one, enhance the ubiquitination level of NLRP3, which might also contribute to the inhibition of NLRP3 inflammasome activation. Finally, BOT-4-one appeared to be superior to other known NLRP3 alkylators in inhibiting the functionality of the NLRP3 inflammasome and its resulting anti-inflammatory activity was confirmed in vivo using a monosodium urate-induced peritonitis mouse model. Collectively, the results suggest that NLRP3 alkylators function by inhibiting ATPase activity and increasing the ubiquitination level of NLRP3, and BOT-4-one could be the type of NLRP3 inhibitor that may be potentially useful for the novel development of a therapeutic agent in controlling NLRP3 inflammasome-related diseases.