Heat shock transcription factor (HSF) mediates the stress-induced expression of heat shock protein genes (hsp). However, HSF is required for normal cell function even in the absence of stress and is important for cell cycle progression, but the mechanism that mediates these effects of HSF is unknown. Here, it is shown that a member of the HSF family, HSF2, interacts with the PR65 (A) subunit of protein phosphatase 2A (PP2A). HSF2 binding to PR65 blocks its interaction with the catalytic subunit, due to competition between HSF2 and catalytic subunit for the same binding site in PR65. In addition, overexpression of HSF2 stimulates PP2A activity in cells, indicating the relevance of HSF2 as a regulator of PP2A in vivo. These results identify HSF2 as a dual function protein, capable of regulating both hsp expression and PP2A activity. This could function as a mechanism by which hsp expression is integrated with the control of cell division or other PP2A-regulated pathways.