Aims: Clinical studies demonstrate attenuation of trigeminal-related pain states such as migraine by intranasal CO2 application. This study investigated the underlying mechanisms of this observation and its potential use to reverse trigeminal pain and hypersensitivity. Main methods: We used a behavioral rat model of capsaicin-induced trigeminal thermal hyperalgesia, intranasal CO2 application and several pharmacologic agents such as carbonic anhydrase, acid-sensing ion channels (ASICs), and TRPV1 blocker as well as acidic buffer solutions to investigate and mimic the underlying mechanism. Key findings: Intranasal CO2 application produced a robust dose-dependent antihyperalgesic effect in rats that lasted at least one hour. Blockade of nasal carbonic anhydrase with a dorzolamide solution (Trusopt® ophthalmic solution) showed only a non-significant decrease of the antihyperalgesic effect of intranasal CO2 application. Pharmacologic blockade of ASICs or TRPV1 receptor significantly attenuated the antihyperalgesic effect of CO2 application. The effect of intranasal CO2 application could be mimicked by application of pH 4, but not pH 5, buffer solution to the nasal mucosa. As with CO2 application, the antihyperalgesic effect of intranasal pH 4 buffer was blocked by nasal application of antagonists to ASICs and TRPV1 receptors. Significance: Our results indicate that intranasal CO2 application results in a subsequent attenuation of trigeminal nociception, mediated by protonic activation of TRPV1 and ASIC channels. A potential central mechanism for this attenuation is discussed. The antihyperalgesic effects of intranasal CO2 application might be useful for the treatment of trigeminal pain states.