NS3 protease plays a vital role in the replication of the hepatitis C virus (HCV). BMS-605339 is a novel linear tetra-peptide alpha-ketoamide inhibitor of NS3 protease and shows specificity for HCV NS3 protease genotype 1a and genotype 1b. Mutation at the key site 168 of the HCV NS3 protease can induce resistance to BMS-605339, which greatly affects the antiviral therapy efficacy to hepatitis C. In the present study, we employed molecular dynamics simulations, free energy calculations, and free energy decomposition to explore the drug resistance mechanism of BMS-605339 due to the three representative mutations D168C/Y/V. The free energy decomposition analysis indicates that the decrease in the binding affinity is mainly attributed to the decrease in both van der Waals and electrostatic interactions. After detailed analysis of our calculated results, we observed that the break of the salt bridge between residues 155 and 168 caused by the mutations D168C/Y/V is the original reason for the decrease in the binding ability between BMS-605339 and the mutant NS3 proteases. The obtained results will reveal the drug resistance mechanism between BMS-605339 and the mutant NS3 proteases, and provide valuable clue for designing novel and more potent drugs to HCV NS3 protease. (C) 2016 International Union of Biochemistry and Molecular Biology, Inc.