Here, the mechanism of vasorelaxant Mas receptor (MasR) expression elevated by hesperidin in spontaneously hypertensive rats was investigated in human umbilical vein endothelial cells (HUVECs). HUVECs were cultured with 1 μM hesperidin for 2 h, following the measurements of nitric oxide (NO) production and vasomotor-related receptors’ expression. Hesperidin significantly promoted NO production (224.1 ± 18.3%, P < 0.01 vs control) in the HUVECs. Only the MasR expression was upregulated (141.2 ± 12.5%, P < 0.05 vs control), whereas a MasR antagonist did not alter the hesperidin-induced NO production. When a transient receptor potential vanilloid 1 (TRPV1) was knocked down by silencing RNA or Ca2+/calmodulin-dependent kinase II (CaMKII) and p38 mitogen-activated protein kinase (p38 MAPK) were inhibited, the increased MasR expression by hesperidin was abrogated. The inhibitions of CaMKII and endothelial NO synthase (eNOS) abolished the hesperidin-induced NO production. The structure–activity relationship analysis of flavonoids demonstrated that the B ring of the twisted flavonoid skeleton with a hydroxy group at the 3′ position was a crucial factor for TRPV1 stimulation. Taken together, it was demonstrated that hesperidin may stimulate TRPV1-mediated cascades, leading to the activation of two signaling axes, CaMKII/p38 MAPK/MasR expression and CaMKII/eNOS/NO production in HUVECs.