Cyclic AMP-response element-binding protein (CREB) plays key transcriptional roles in cell metabolism, proliferation, and survival. Ser133 phosphorylation by protein kinase A (PKA) is a well-characterized CREB activation mechanism. Homeodomain-interacting protein kinase (HIPK) 2, a nuclear serine/threonine kinase, activates CREB through Ser271 phosphorylation; however, the regulatory mechanism remains uncharacterized. Transfection of CREB in HEK293 cells together with the kinase demonstrated that HIPK2 phosphorylated CREB at Ser271 but not Ser133; likewise, PKA phosphorylated CREB at Ser133 but not Ser271, suggesting two distinct CREB regulatory mechanisms by HIPK2 and PKA. In vitro kinase assay revealed that HIPK2, and HIPK1 and HIPK3, directly phosphorylated CREB. Cells exposed to 10μM sodium arsenite increased the stability of HIPK1 and HIPK2 proteins, leading to CREB activation via Ser271 phosphorylation. Phospho-Ser271 CREB showed facilitated interaction with the TFIID subunit coactivator TAF4 assessed by immunoprecipitation. Furthermore, a focused gene array between cells transfected with CREB alone and CREB plus HIPK2 over empty vector-transfected control displayed 14- and 32-fold upregulation of cyclin A1, respectively, while no upregulation was displayed by HIPK2 alone. These results suggest that the HIPK2-phospho-Ser271 CREB axis is a new arsenic-responsive CREB activation mechanism in parallel with the PKA-phospho-Ser133 CREB axis.