Obesity is a well-known public health issue around the world. Sepsis is a lethal clinical syndrome that causes multiorgan failure. Obesity may aggravate inflammation in septic patients. Glutamine (GLN) is a nutrient with immune regulatory and anti-inflammatory properties. Since sepsis is a common contributing factor for acute kidney injury (AKI), this study investigated the effects of GLN administration on sepsis-induced inflammation and AKI in obese mice. A high-fat diet which consists of 60% of calories from fat was provided for 10 weeks to induce obesity in the mice. Then, the obese mice were subdivided into sepsis with saline (SS) or GLN (SG) groups. Cecal ligation and puncture (CLP) was performed to produce sepsis. The SS group was intravenously injected with saline while the SG group was administered GLN one or two doses after CLP. Obese mice with sepsis were sacrificed at 12, 24, or 48 h post-CLP. Results revealed that sepsis resulted in upregulated high-mobility group box protein-1 pathway-associated gene expression in obese mice. Also, expressions of macrophage/neutrophil infiltration markers and inflammatory cytokines in kidneys were elevated. Obese mice treated with GLN after sepsis reversed the depletion of plasma GLN, reduced production of lipid peroxides, and downregulated macrophage/neutrophil infiltration and the inflammatory-associated pathway whereas tight junction gene expression increased in the kidneys. These findings suggest that intravenously administered GLN to obese mice after sepsis alleviated inflammation and attenuated AKI. This model may have clinical application to obese patients with a risk for infection in abdominal surgery.