Transient receptor potential vanilloid 4 (TRPV4) channel is known to regulate vascular tone in the mesenteric artery and the pulmonary artery. We investigated the effect of GSK1016790A, a TRPV4 channel activator, on the retinal arteriolar diameter and the underlying mechanisms in rats. The retinal arteriolar diameters were measured using the ocular fundus images captured with a high-resolution digital camera in vivo. The retinal arteriolar diameter was increased by intravenous infusion of GSK1016790A in a dose-dependent manner. The mean arterial pressure was slightly decreased by infusion of a higher dose of GSK1016790A. Intravenous injection of GSK2193874, a TRPV4 channel blocker, significantly reduced the dilator effects of GSK1016790A on retinal arterioles. Intravitreal injection of N^ω-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, and iberiotoxin, a large-conductance Ca²⁺-activated K⁺ channel blocker, significantly attenuated the GSK1016790A-induced dilation of retinal arterioles. The inhibitory effect of L-NAME and iberiotoxin was additive. The results in the present study suggest that activation of TRPV4 channels dilates the rat retinal arterioles via the production and release of nitric oxide and the activation of large-conductance Ca²⁺-activated K⁺ channels in vivo.