Abstract Background Transient receptor potential melastatin 2 is a Ca2+-permeable cation channel abundantly expressed in macrophages. Trpm2 −/− mice showed exacerbated infection and mortality during polymicrobial sepsis, which is associated with inefficient bacterial killing in macrophages. However, the mechanism of transient receptor potential melastatin 2 regulating bacterial killing remains unknown. Methods Trpm2 −/− mice were intraperitoneally injected with Escherichia coli. The survival rate (n = 21) and bacterial burden (n = 5) were assessed. The processes of phagosome maturation and phagosome–lysosome fusion in peritoneal macrophages were extensively studied. The impact of increasing intracellular Ca2+ concentration on bacterial clearance in macrophages (n = 3) and on survival rate of Trpm2 −/− mice infected with E. coli (n = 21) was investigated. Results Trpm2 −/− mice exhibited increased mortality (85% vs. 54%; P < 0.01) and aggravated bacterial burden during E. coli sepsis. Trpm2 −/− peritoneal macrophages infected with E. coli showed dampened recruitment of lysosomal-associated membrane protein 1 and impaired phagosome maturation evidenced by a decrease in the accumulation of early endosome antigen 1, whereas a normal acquisition of Ras-related protein in brain 5. Increasing the cytosolic Ca2+ concentration in Trpm2 −/− peritoneal macrophages via ionomycin treatment facilitated early endosome antigen 1 recruitment to Ras-related protein in brain 5 and phagosomal localization of lysosomal-associated membrane protein 1 and consequently enhanced bactericidal activity. Adoptive transfer of ionomycin-treated Trpm2 −/− peritoneal macrophages improved bacterial clearance and survival (67% vs. 29%; P < 0.01) in Trpm2 −/− mice challenged with E. coli. Conclusions Transient receptor potential melastatin 2 plays a critical role in host defense against invading bacteria via promoting phagosome maturation through facilitation of early endosome antigen 1 recruitment.