The nuclear oncoprotein prothymosin α (ProTα) was tested for the ability to regulate the p53 activity with the use of a reporter gene controlled by a p53-responsive promoter. Overexpression of the ProTα gene stimulated the p53 activity, while downregulation of the endogenous ProTα level via RNA interference suppressed transcription of the reporter gene. An increase in ProTα activated p53-dependent transcription and increased the intracellular p53 content in human HeLa, but not HCT116, cells. N-terminal deletions had almost no effect on the ability of ProTα to activate p53-dependent transcription, while deletions from the central region and C-terminal mutations distorting the active transport of ProTα into the cell nucleus prevented its transactivating effect. Mutations affecting Keap1 binding did not impair the ProTα ability to activate the p53-responsive reporter gene. Based on the results, stimulation of p53-dependent transcription was ascribed to the central acidic region of ProTα. The conclusion was supported by the fact that parathymosin, another protein containing an extended acidic region, was also capable of activating p53.