Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR2) than on HIV-1 protease (PR1). We solved the crystal structure of PR2 with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR1 mutant (PR1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR2. PR1M more closely resembled PR2 than PR1 in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR1M with APV, DRV, and SQV were compared with available PR1 and PR2 complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR1M and PR1, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR1M and PR2 relative to the strong hydrogen bonds observed in PR1, consistent with 15- and 19-fold weaker inhibition, respectively. Overall, PR1M partially replicates the specificity of PR2 and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2.