Down-regulation of Kv4.3 K⁺ channels commonly occurs in multiple diseases, but the understanding of the regulation of Kv4.3 K⁺ channels and the role of Kv4.3 K⁺ channels in pathological conditions are limited. HEK (human embryonic kidney)-293T cells are derived from HEK-293 cells which are transformed by expression of the large T-antigen. In the present study, by comparing HEK-293 and HEK-293T cells, we find that HEK-293T cells express more Kv4.3 K⁺ channels and more transcription factor Sp1 (specificity protein 1) than HEK-293 cells. Inhibition of Sp1 with Sp1 decoy oligonucleotide reduces Kv4.3 K⁺ channel expression in HEK-293T cells. Transfection of pN3-Sp1FL vector increases Sp1 protein expression and results in increased Kv4.3 K⁺ expression in HEK-293 cells. Since the ultimate determinant of the phenotype difference between HEK-293 and HEK-293T cells is the large T-antigen, we conclude that the large T-antigen up-regulates Kv4.3 K⁺ channel expression through an increase in Sp1. In both HEK-293 and HEK-293T cells, inhibition of Kv4.3 K⁺ channels with 4-AP (4-aminopyridine) or Kv4.3 small interfering RNA induces cell apoptosis and necrosis, which are completely rescued by the specific CaMKII (calcium/calmodulin-dependent protein kinase II) inhibitor KN-93, suggesting that Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through CaMKII activation. In summary, we establish: (i) the HEK-293 and HEK-293T cell model for Kv4.3 K⁺ channel study; (ii) that large T-antigen up-regulates Kv4.3 K⁺ channels through increasing Sp1 levels; and (iii) that Kv4.3 K⁺ channels contribute to cell apoptosis and necrosis through activating CaMKII. The present study provides deep insights into the mechanism of the regulation of Kv4.3 K⁺ channels and the role of Kv4.3 K⁺ channels in cell death.