Colorectal cancer (CRC) is among the leading causes of cancer-related death throughout the world. Aberrant expression of voltage-gated potassium ion channel molecule Kv1. 3 has been reported in various cancers. To explore the expression and regulation of Kv1. 3 in colorectal cancer, 7 colorectal cancer cell lines and 147 cases of primary colorectal cancer were involved in this study. Kv1. 3 was expressed in LOVO and SW480 cells and loss of expression was found in RKO, DLD1, SW620, HCT116, and HT29 cells. Complete methylation was found in RKO, DLD1, SW620, HCT116, and HT29 cells, partial methylation was found in LOVO and SW480 cells. Loss of/reduced expression of Kv1. 3 is correlated with methylation of its promoter region. The expression of Kv1. 3 was restored in RKO, DLD1, SW620, HCT116, and HT29 cells, and increased in LOVO and SW480 cells after 5-aza-2'-deoxycytidine (DAC) treatment. The results suggest that the expression of Kv1. 3 is regulated by the gene's promoter region methylation in human CRC cells. Kv1. 3 was methylated in 76.19%(112/147) of primary human colorectal cancer. Methylation of Kv1. 3 is associated with age, tumor differentiation, and poor 5-year survival. In conclusion, Kv1. 3 is frequently methylated in human colorectal cancer and the expression of Kv1. 3 is regulated by its promoter region methylation. Kv1. 3 methylation may serve as diagnostic and prognostic markers in colorectal cancer.