As a member of purinoceptors, the P2Y6 receptor (P2Y6R) plays a crucial role in modulating immune signals and has been considered as a potential therapeutic target for inflammatory diseases. On the basis of the speculated probable conformation and binding determinants of P2Y6R, a hierarchical strategy that combines virtual screening, bioassays, and chemical optimization was presented. A potent P2Y6R antagonist (compound 50) was identified to possess excellent antagonistic activity (IC50 = 5.914 nM) and high selectivity. In addition, binding assays and chemical pull-down experiments confirmed that compound 50 was nicely bound to P2Y6R. Notably, compound 50 could effectively ameliorate DSS-induced ulcerative colitis in mice through inhibiting the activation of NLRP3 inflammasome in colon tissues. Moreover, treatment with compound 50 reduced LPS-induced pulmonary edema and infiltration of inflammatory cells in mice. These findings suggest that compound 50 could serve as a specific P2Y6R antagonist for treating inflammatory diseases and deserve further optimization studies.