Steroid receptor coactivator 3 (SRC-3) is a multifunctional protein that plays an important role in regulation of bacterial lipopolysaccharide-induced inflammation. However, its involvement in host defense against bacterial infection remains unclear. Herein we used SRC-3 knockout mice to assess the role of SRC-3 in antibacterial defense in E. coli-induced septic peritonitis. After E. coli bacteria were injected intraperitoneally, SRC-3 deficient mice exhibited excessive local and systemic inflammatory responses and more severe bacterial burdens, leading to a significantly higher mortality compared with wild-type mice. Peritoneal macrophages of SRC-3 deficient mice showed a decrease in bacterial phagocytosis in culture and an increase in apoptosis, which was consistent with the defective bacterial clearance observed in SRC-3-deficient mice. Accordingly, SRC-3 null macrophages expressed much lower levels of the scavenger receptor A, the antioxidant enzyme catalase, and anti-apoptotic gene Bcl-2. Collectively, our data demonstrate that SRC-3 is important in not only modulating the local and systemic inflammation but also intensifying bacterial clearance, which highlights a pivotal role of SRC-3 in the host defense system against bacterial infection.