Exposure to hyperbaric oxygen has been shown to the effective in cluster headache, but the mechanism of the action is still not clear. Primary nociceptive neurons, containing neuropeptides such as substance P and particularly those innervating the nasal mucosa, could be involved in the pathogenesis of cluster headache. The present study evaluated the effect of an exposure to hyperbaric oxygen on the content of substance P in the nasal mucosa of patients affected by cluster headache. The results were compared with those observed in another group of cluster headache patients who underwent a placebo procedure. The samples of nasal mucosa were analyzed by immunocytochemical methods. A qualitative analysis of the slides was carried out by an operator under “blinded conditions.” A marked decrease in the content of immunoreactivity for substance P was found in the patients exposed to hyperbaric oxygen. The decrease was statistically significant when compared with the findings of the placebo procedure. The results of the present study indicate that an influence on the content of peripheral neuropeptides could be involved in the mechanism of action of the beneficial effect of hyperbaric oxygen in cluster headache.