Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.