Sepsis-associated acute kidney injury (AKI) is a serious clinical problem, without effective drugs. Abnormal activation of the purinergic P2X7 receptor (P2X7R) in septic kidneys makes its antagonist a promising therapeutic approach. Herein, a series of novel P2X7R antagonists were designed, synthesized, and structurally optimized. Based on in vitro potency in human/mouse P2X7R using HEK293 cells, hepatic microsomal stability, and pharmacokinetic and preliminary in vivo assessments, compound 14a was identified by respective human and mouse P2X7R IC50 values of 64.7 and 10.1 nM, together with favorable pharmacokinetic properties. Importantly, 14a dose-dependently alleviated kidney dysfunction and pathological injury in both lipopolysaccharide (LPS)- and cecal ligation/perforation (CLP)-induced septic AKI mice with a good safety profile. Mechanistically, 14a could suppress NLRP3 inflammasome activation to inhibit the expression of cleaved caspase-1, gasdermin D, IL-1β, and IL-18 in the injured kidneys of septic mice. Collectively, these results highlighted that P2X7R antagonist 14a exerted a therapeutic potential against septic AKI.