Abstract: In the study, effects of temperature and time on the inactivation of microorganisms of fresh litchi juice by dimethyl carbonate (DMDC) were investigated, and the changes in the counts of microorganisms of DMDC-treated fresh litchi juice during storage at 4were also determined. Results showed that the inactivation rate of microorganisms increased with the increasing temperature. But the increase of the temperature could not increase the inactivation of microorganisms, which only decreased the inactivation time of microorganisms. After adding 250 mg/L of DMDC for 12 h, a complete inactivation of yeasts was observed, and the counts of total bacteria, lactic acid bacteria and mold declined to 3.73, 2.6 and 0.3 lg (CFU/mL), respectively. The identification using 16S or 18S rDNA methods found the residual microorganisms in DMDC-treated litchi juice mainly were Bacillus, Mucor and Leuconostoc strains, and the Leuconostoc was the dominant strains. The residual Leuconostoc strains in DMDC-treated litchi juice showed growth during storage of 4益, after 40 d of storage, the count of Leuconostoc strains reached more than 5 lg, which impacted the nutritional quality and flavor of litchi juice.