Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase (eNOS-Tg) have a 20% lower systemic vascular resistance (SVR) than wild type (Wt) mice. However, as eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by differences in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg compared to Wt mice, consistent with increased NO synthesis. Vasodilation to SNP was reduced whereas the vasodilator responses to phosphodiesterase-5 blockade and 8Br-cGMP were maintained in eNOS-Tg compared to Wt mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, as scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, while after eNOS-inhibition the vasodilator response to ROS-scavenging was similar in Wt and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone (including cyclooxygenase, cytochrome P450 2C9, endothelin, adenosine, and K⁺_Ca-channels) did not significantly affect SVR in either eNOS-Tg or Wt mice, while the marked vasoconstrictor responses to K⁺_ATP- and K⁺_V-channel blockade were similar in Wt and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.