Capsaicin bestows spiciness by activating TRPV1 channel with exquisite potency and selectivity. Although a capsaicin-bound channel structure was previously resolved by cryo-EM at 4.2-to 4.5-Å resolution, capsaicin was registered as a small electron density, reflecting neither its chemical structure nor specific ligand-channel interactions—important details required for mechanistic understanding. We obtained the missing atomic-level details by iterative computation and confirmed them by systematic site-specific functional tests. We observed that the bound capsaicin takes a'tail-up, head-down'configuration. The vanillyl and amide groups form specific interactions to anchor its bound position, while the aliphatic tail may sample a range of conformations, making it invisible in cryo-EM images. Capsaicin stabilizes TRPV1's open state by'pull-and-contact'interactions between the vanillyl group and the S4-S5 linker. Our study provides a structural mechanism for the agonistic function of capsaicin and its analogs, and demonstrates an effective approach to obtain atomic-level information from cryo-EM structures.