Regulation of cytoplasmic p21CIP/WAF1 (p21) is of great clinical significance in molecular oncology due to its identification as an antiapoptotic factor, a poor survival predictor as well as a drug-resistance inducer. A retrospective study of the immunohistochemical (IHC) profiles of 128 human primary breast cancers showed that increased total and cytoplasmic p21 expression were highly associated with the expression of IkappaB kinase beta (IKKbeta), the major catalytic subunit of the IKK complex and another crucial player in tumorigenesis and drug resistance. The causal relationship study based on cultured cell lines, MDA-MB-453 and MCF-7, confirmed that IKKbeta overexpression did upregulate protein levels of total and cytoplasmic p21. Mechanistic investigation demonstrated that IKKbeta increased p21 expression through upregulation of p21 mRNA level. Moreover, by Western blotting, IKKbeta was found to be able to upregulate Akt phosphorylation on Ser 473. This novel finding indicated that IKKbeta could mediate cytoplasmic p21 accumulation via activation of its downstream target Akt, which was known to phosphorylate p21 and lead to cytoplasmic localization of p21.