Acute kidney injury (AKI) is a rapid loss of kidney function characterized by damage to renal tubular cells driven by mitochondrial dysregulation and oxidative stress. Here, we used a murine caecal ligation and puncture (CLP) model of sepsis-induced AKI to study the role of sirtuin 3 (SIRT3), a NAD⁺ dependent deacetylase critical for the maintenance of mitochondrial viability, in AKI-related renal tubular cell damage and explored the underlying mechanisms. CLP induced alterations in kidney function and morphology were associated with SIRT3 downregulation, and SIRT3 deletion exacerbated CLP-induced kidney dysfunction, renal tubular cell injury and apoptosis, mitochondrial alterations, and ROS production in a knockout mouse model. SIRT3 deletion increased the CLP-induced upregulation of the NLRP3 inflammasome and apoptosis-associated speck-like protein, resulting in the activation of oxidative stress, increased production of the proinflammatory cytokines interleukin (IL)-1β and IL-18, and the enhancement of apoptosis, and these effects were reversed by antioxidant NAC. Our results suggest that SIRT3 plays a protective role against mitochondrial damage in the kidney by attenuating ROS production, inhibiting the NRLP3 inflammasome, attenuating oxidative stress, and downregulating IL-1β and IL-18.