Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.