Elastase released by neutrophils is critical for eliminating Gram-negative bacteria. Ca2+ influx plays a key role in elastase release and bacterial clearance in neutrophils. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable cation channel highly expressed in neutrophils. Here, we explore the role and possible mechanism of TRPM2 in bacterial clearance in TRPM2 knockout (TRPM2-KO) mice neutrophils. After exposure to Escherichia coli, TRPM2–KO bone marrow neutrophils (BMNs) had increased bacterial burden and decreased elastase release. The same was observed for septic TRPM2-KO mice which also had decreased survival rate. After stimulation with chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), elastase release was lower in TRPM2-KO BMNs than in wild type (WT) BMNs. Pre-treatment of WT BMNs with p38 MAPK inhibitor reduced fMLP-induced elastase release. Compared with WT BMNs, TRPM2-KO BMNs had decreased p38 MAPK phosphorylation after fMLP stimulation. Removal of extracellular Ca2+ reduced fMLP-induced p38 MAPK phosphorylation and elastase release. The concentration of intracellular Ca2+ decreased in TRPM2-KO BMNs compared with WT BMNs after fMLP treatment. Hence, TRPM2 plays an important role in bacterial clearance in neutrophils, possibly by regulating elastase release. TRPM2-mediated Ca2+ influx regulates elastase release partially via p38 MAPK phosphorylation in neutrophils.