Aim To investigate the role of calcineurin/nuclear factor of activated T cells (CaN/NFAT) signaling pathway in aerobic exercise-induced increased expression of KV2.1 channel in mesenteric artery vascular smooth muscle cell from spontaneously hypertensive rats (SHR). Methods Male SHR and Wistar-Kyoto rats (WKY), 12 weeks age, were randomly divided into control groups (WKY-SED, SHR-SED), and exercise groups (WKY-EX, SHR-EX). Exercise groups were subjected to a 12-week treadmill training protocol:20 m/min, 60 min/day, 5 d/w. After 12 weeks, heart rate (HR) and blood pressure (BP) was acquired in each group. Immunocytochemistry was used to observe the distribution and changing of KV2.1 and CaN in mesenteric artery (MA). Western blot was applied to examine the expression levels of KV2.1, CaN, NFATc3, p-NFATc3, A-kinase anchoring protein (AKAP150) and calmodulin (CaM) protein in MA. Results (1)After 12 weeks of exercise, HR, systolic blood pressure (SBP)and mean blood pressure (MBP) in SHR-EX group was significantly lower than that in SHR-SED group (P＜0.01); SBP in WKY-EX group was lower than that in WKY-SED group (P＜0.05). (2) The distribution and protein expression of KV2.1 in SHR-SED group was significantly lower than that in WKY-SED group (P＜0.01); Exercise training markedly increased the expression of KV2.1 in SHR-EX group (P＜0.01). (3) Compared with the WKY-SED group, the distribution and protein expression of CaN was significantly increased, but the protein expression of p-NFATc3 was significantly lower in SHR-SED group(P＜0.01); Exercise training markedly inhibited the distribution and expression of CaN and increased the expression of p-NFATc3 in SHR-EX group (P＜0.01). (4) The protein expression of AKAP150 in SHR-SED group was significantly higher than that of WKY-SED group (P＜0.01); aerobic exercise obviously decreased the upregulation protein expression of AKAP150 in hypertension(P＜0.01). (5) The protein expression of CaM in SHR-SED group was significantly higher than that of WKY-SED group (P＜0.01); Compared with the SHR-SED group, the protein expression of CaM was significantly decreased in SHR-EX group (P＜0.01); Compared with the WKY-SED group, the protein expression of CaM was significantly increased in WKY-EX group (P＜0.05). Conclusion The increased CaN/NFAT signaling pathway may induce the KV2.1 expression downregulation, however, aerobic exercise can effectively inhibit such effects, which may be the mechanism of exercise restoring cardiovascular function.