Transient receptor potential vanilloid type 1 (TRPV1) channels are activated by heat, vanilloids, and extracellular protons. Cryo-EM has revealed various conformations of TRPV1, and these structures suggest an intramolecular twisting motion in response to ligand binding. However, limited experimental data support this observation. Here, we analyzed the intramolecular motion of TRPV1 using diffracted X-ray tracking (DXT). DXT analyzes trajectories of Laue spots generated from attached gold nanocrystals and provides picometer spatial and microsecond time scale information about the intramolecular motion. We observed that both an agonist and a competitive antagonist evoked a rotating bias in TRPV1, though these biases were in opposing directions. Furthermore, the rotational bias generated by capsaicin was reversed between the wild-type and the capsaicin-insensitive Y511A mutant. Our findings bolster the understanding of the mechanisms used for activation and modulation of TRP channels, and this knowledge can be exploited for pharmacological usage such as inhibitor design.