Objective：To investigate the efficacy and mechanism of baicalin（BA）inhibiting coxsackie virus B3（CVB3） infection in vitro. Methods：Hela cell viability was measured by CellTiter 96- AQueous single reagent. The virus titer was detected by virus plaque assay. The concentration of cholesterol（CHO）and free fatty acids（FAA）were detected by CHOD-PAP and ACS-ACOD，respectively. The expression levels of fatty acid synthase（Fasn） mRNA and acetyl-CoA carboxylase（ACC） mRNA were detected by RT-PCR. The expression of VP1 protein was detected by Western blot in CVB3 virus. Results：BA had no significant toxic effect on Hela cells at less than 100 μg/mL，but was able to against cytopathy by CVB3 in a dose-dependent manner. The viral titers and CVB3/VP1 expression were significantly reduced by BA treatment in CVB3 infected cells. The levels of CHO and FAA，mRNA of Fasn and ACC were all significantly expressed in CVB3 infected cells，but ware all significantly reduced by BA treatment. Compared with BA treatment group by BSA pretreatment，palmitic acid pretreatment could increase the expression of CVB3/VP1 in Hela cells. Conclusion：BA can against CVB3 infection by regulating cellular lipid synthesis.