Affinin is mainly recognized by its antinociceptive effect. Recently, our research group demonstrated that this compound produces vasodilation via activation of the gasotransmitters signaling pathways. However, the molecular targets of affinin were not identified. Considering the structural similarity of this alkamide with anandamide, we hypothesized that affinin-induced vasodilation could involve participation of TRP channels and cannabinoid receptors. In this work, by using the isolated rat aorta assay, we assessed involvement of TRP channels, the cannabinoid system, and the HNO-CGRP-TRPA 1 pathway on the mechanism of action of affinin. Additionally, we measured NO and H 2 S levels elicited by affinin on rat aorta homogenates and carried out computer simulations of molecular interactions between affinin and the TRPA 1 and TRPV 1 channels and the CB 1 receptor. Our results indicated that affinin induces an increase in aortic NO and H 2 S levels. We found evidence that the vasodilator effect induced by affinin involves activation of TRPA 1 and TRPV 1 channels and the CB 1 and eCB receptors. In silico analyses showed that affinin is able to bind with high affinity to these molecular targets. Moreover, we also proved that affinin-induced vasodilation is partly mediated via activation of the HNO-TRPA 1 -CGRP pathway. Based on these results we propose a novel mechanism of action to explain the vasodilatory effect of affinin, which could be developed as an alternative drug to treat cardiovascular diseases. [Display omitted]