TRPV1 ion channel is a prototypical nociceptor. Vanilloids with similar structures, such as capsaicin, resiniferatoxin (RTX), and capsazepine, exhibit highly variable gating effects, from being a potent agonist to being an antagonist. To understand the structural and mechanistic differences in gating between these vanilloids, we first isolated intermediates states with varying number of bound RTX, using concatemers of wildtype rat TRPV1 subunits (RTX binds irreversibly) and Y511A mutant subunits (RTX binds reversibly). Equilibrium measurements from these concatemers revealed that binding of RTX to each subunit contributes 1.8 kcal/mol towards stabilizing the open state. Replacing RTX with capsaicin (whose binding to wildtype or mutant subunit is reversible) revealed that the Y511A mutation reduced capsaicin binding affinity by two orders of magnitude; capsaicin exerts a similar stabilization energy as RTX. Finally, we compared the energetic differences between RTX and two derivatives (5’-iRTX and 6’-iRTX), in which a one-atom difference makes the analogous molecules exhibit completely different gating effects on TRPV1: RTX is a full agonist, 6’-iRTX is a partial agonist, and 5’iRTX is an antagonist. We found that compared with RTX, 6’-iRTX only makes a small contribution (0.3 kcal/mol) to stabilize the open state, while 5’-iRTX contributes 1.2 kcal/mol to stabilize the close state. Combining energetic information with computational modeling and structural details of ligand-channel interactions allows a better understanding of TRPV1 activation and nociception.