Capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) is activated by various noxious stimuli, and the stimuli are converted to electrical signals in primary sensory neurons. It is believed that cation influx through TRPV1 causes depolarization, leading to the activation of voltage-gated sodium channels, followed by action potential generation (1). Anoctamin 1 (ANO1), a calcium-activated chloride channel, is also expressed in the TRPV1-positive DRG neurons (2). Therefore, we hypothesized that calcium entering the cells through TRPV1 activation induces ANO1 activation followed by further depolarization in DRG neurons because the intracellular chloride concentrations are maintained at a high level due to low expression of potassium-chloride co-transporter type 2 (3). We suggest the pain enhancement mechanism through the interaction between TRPV1 and ANO1 in DRG neurons. Capsaicin-activated currents were significantly larger in HEK293T cells expressing both TRPV1 and ANO1 than in cells expressing TRPV1 or ANO1 alone. Furthermore, direct interaction between TRPV1 and ANO1 was suggested by immunoprecipitation in both HEK293T cells and DRG, which could effectively drive the TRPV1-ANO1 functional interaction through the increase in intracellular calcium. In addition, in mouse DRG neurons, capsaicin-activated inward currents were significantly inhibited by a specific ANO1 antagonist, T16Ainh-A01 (A01) in the presence of a high concentration of EGTA, but not BAPTA. And the concomitant administration of A01 inhibited capsaicin-evoked action potential generation in DRG neurons probably through blocking the interaction because A01 did not affect the action potential generations by current injection and the currents of voltage-gated sodium, potassium and calcium channels. Furthermore, capsaicin-evoked pain-related behaviors were inhibited by A01. TRPV1 and ANO1 work as receptors activated by noxious stimuli in sensory nerve endings. It is believed that activation of the two channels causes cation influx and anion efflux, respectively, both of which lead to depolarization. We show that ANO1 is activated by calcium ions entering neurons through TRPV1 activation based on their physical binding on the cell membrane. Indeed, both capsaicin-activated inward currents in sensory neurons and capsaicin-induced pain-related behaviors in mice were significantly inhibited by ANO1 blockade. Thus, the interaction between TRPV1 and ANO1 functions is a pain-enhancing mechanism.