Emerging epidemiological and experimental evidence has shown that the ATP2B1 gene is associated with blood pressure control. Impaired eNOS activity and NO production may be among the mechanisms involved. However, little is known about how PMCA1, which is encoded by the ATP2B1 gene, regulates the activity of eNOS and NO production. In the present study, we investigated the role of the ATP2B1 gene in regulating eNOS activity and NO production under basal conditions in HUVECs and explored the mechanisms involved. Silencing ATP2B1 gene expression resulted in higher NO production and eNOS activity under basal conditions in HUVECs. Additionally, ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations compared to that in the negative siRNA-transfected HUVECs. The enhanced eNOS activity mediated by ATP2B1 gene silencing was Ca2+/calmodulin dependent, as verified by the administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Taken together, silencing ATP2B1 gene expression results in higher NO production and eNOS activity under basal conditions in HUVECs. Furthermore, the enhanced eNOS activity induced by ATP2B1 gene silencing may be mediated via higher levels of intracellular Ca2+, and the effect was confirmed to be dependent on the eNOS-calmodulin interaction.