Bortezomib is a proteasome inhibitor approved for anticancer therapy. However, variable sensitivity of tumor cells exists in this therapy probably due to differences in the expression of proteasome subunits. G(alpha)(12/13) serves modulators or signal transducers in diverse pathways. This study investigated whether cancer cells display differential sensitivity to bortezomib with reference to G(alpha)(12/13) expression, and if so, whether G(alpha)(12/13) affects the expression of proteasome subunits and their activities. Bortezomib treatment exhibited greater sensitivities in Huh7 and SNU886 cells (epithelial type) than SK-Hep1 and SNU449 cells (mesenchymal type) that exhibited higher levels of G(alpha)(12/13). Overexpression of an active mutant of G(alpha)(12) (Galpha(12)QL) or G(alpha)(13) (G(alpha)(13)QL) diminished the ability of bortezomib to induce cytotoxicity in Huh7 cells. Moreover, transfection with the minigene that disturbs G protein-coupled receptor-G protein coupling (CT12 or CT13) increased it in SK-Hep1 cells. Consistently, MiaPaCa2 cells transfected with CT12 or CT13 exhibited a greater sensitivity to bortezomib. Evidence of G(alpha)(12/13)'s antagonism on the anticancer effect of bortezomib was verified in the reversal by G(alpha)(12)QL or G(alpha)(13)QL of the minigenes' enhancement of cytotoxity. Real-time polymerase chain reaction assay enabled us to identify PSMB5, multicatalytic endopeptidase complex-like-1, and proteasome activator subunit-1 repression by CT12 or CT13. Furthermore, G(alpha)(12/13) inhibition enhanced the ability of bortezomib to repress PSMB5, as shown by immunoblotting and proteasome activity assay. Moreover, this inhibitory effect on PSMB5 was attenuated by G(alpha)G(alpha)(12)QL or G(alpha)(13)QL. In conclusion, the inhibition of G(alpha)(12/13) activities may enhance the anticancer effect of bortezomib through PSMB5 repression, providing insight into the G(alpha)(12/13) pathway for the regulation of proteasomal activity.