This study investigated how a natural sphalerite (NS) photocatalyst, under visible light irradiation, supports photocatalytic bacterial inactivation. This was done by comparing parent E. coli BW25113, and its two isogenic single-gene knock-out mutants, E. coli JW0797-1 (dps– mutant) and JW1721-1 (katE– mutant), where both dps and KatE genes are likely related to H2O2 production. NS could inactivate approximately 5-, 7- and 7-log of E. coli BW25113, JW0797-1, and JW1721-1 within 6 h irradiation, respectively. The two isogenic mutants were more susceptible to photocatalysis than the parental strain because of their lack of a defense system against H2O2 oxidative stress. The ability of in situ resultant H2O2 to serve as a defense against photocatalytic inactivation was also confirmed using scavenging experiments and partition system experiments. Studying catalase activity further revealed that in situ H2O2 played an important role in these inactivation processes. The destruction of bacterial cells from the cell envelope to the intracellular components was also observed using field emission-scanning electron microscopy. Moreover, FT-IR was used to monitor bacterial cell decomposition, key functional group evolution, and bacterial cell structures. This is the first study to investigate the photocatalytic inactivation mechanism of E. coli using single-gene deletion mutants under visible light irradiation.