While brain-derived neurotrophic factor (BDNF) was previously reported to induce relaxation of conduit artery, whether the BDNF/TrkB (tropomyosin-related kinase) pathway is involved in the tone control of resistance arteries is not known. This study investigated TrkB receptors levels/localization and the vasomotor effect of the TrkB receptor agonist LM22A-4 in isolated third-order mesenteric arteries from rats. Immunostaining revealed the presence of both full-length and truncated TrkB receptors, especially at the endothelial level. By using wire myography, LM22A-4 induced vascular relaxation that was significantly decreased by cyclotraxin B as a non-competitive TrkB antagonist and fully prevented by endothelium removal. Inhibitors of NO, EDHF, PGI2 production and the PI3K/Akt pathways separately reduced LM22A-4 induced-relaxation. By contrast, inhibition of Raf/MEK, PLCγ and CaM/CaMKII pathways did not change the relaxant effect of LM22A-4. Interestingly, BDNF also induced an endothelium and TrkB-dependent relaxation. These results indicate that endothelial TrkB activation results in the relaxation of resistance vessels via PI3K/Akt-induced eNOS phosphorylation and production of EDHF and PGI2. These data are consistent with the contribution of the endothelial BDNF/TrkB pathway to the regulation of peripheral vascular tone. They also validate the use of LM22A-4 as a reliable pharmacological agent for studying the vascular effect of BDNF.