Proteasome inhibitors such as bortezomib constitute novel therapeutic agents that are currently in clinical use and in clinical trials. In some neoplasms, cyclin-dependent kinase inhibitors (CKI) such as p21(WAF1) have been proposed as key targets of proteasome inhibitors. p21(WAF1) expression can be modulated by p53, a tumor suppressor, and especially in breast cancer cells, by estrogen receptor alpha (ERα), which is highly relevant to cancer growth. We investigated the effects of bortezomib using a panel of six cancer cell lines with variable status of ERα or p53 and found that bortezomib inhibited the growth of all cell lines in the same concentration range irrespective of the ERα expression or the mutational status of p53. Bortezomib treatment significantly enhanced p21(WAF1) protein levels in all cell lines but with different mechanisms according to ERα status. In ERα-positive cells, bortezomib treatment caused a strong increase in p21(WAF1) mRNA, whereas in ERα-negative cells it predominantly enhanced p21(WAF1) protein levels suggesting a posttranslational mechanism of p21(WAF1) regulation in the ERα-negative cells. Moreover, the antiproliferative activity of bortezomib was prevented by ERα silencing or p21(WAF1) knockdown in ERα-positive cells. Collectively, our results highlight the potential roles of ERα and p21(WAF1) in growth inhibition of cancer cells mediated by proteasome inhibitors, such as bortezomib.