The mechanisms of anti-microbial photodynamic technology(APDT) against Gram-negative bacteria Escherichia coli O157 by methylene blue(MB) was investigated.The bactericidal effect of MB-APDT on E.coli O157 was measured by counting the reduction of colony forming unit(CFU).The damage of DNA was observed by real-time polymerase chain reaction(RT-PCR) and the degradation of proteins was investigated by sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE).Results showed that almost all of the E.coli O157 were photo inactivated by illumination with 30 min visible light(power density 200 mW/cm2) in the presence of 10 mg/L MB.The significant photo-inactivation of Gram-negative bacteria Escherichia coli O157 may be due to the damage of DNAs and the degradation of proteins.