Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate–cytochrome c reductase (SCR) with Ki = 0.033 ± 0.00027 nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki = 12.90 ± 0.91 μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2. Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.