We previously suggested that therapeutic effects of betahistine in vestibular disorders result from its antagonist properties at histamine H3 receptors (H3Rs). However, H3Rs exhibit constitutive activity and most H3R antagonists act as inverse agonists. Here, we have first investigated the effects of betahistine at recombinant H3R isoforms. On inhibition of cAMP formation and [3H]arachidonic acid release, betahistine behaved as a nanomolar inverse agonist and a micromolar agonist. Both effects were suppressed by pertussis toxin, were found at all isoforms tested, and were not detected in mock cells, confirming interactions at H3Rs. The inverse agonist potency of betahistine and its affinity on [125I]iodoproxyfan binding were similar in rat and human. We have then investigated the effects of betahistine on histamine neuron activity, by measuring tele-methylhistamine (t-MeHA) levels in the brain of mice. Its acute intraperitoneal administration increased t-MeHA levels with an ED50 of 0.4 mg/kg, indicating inverse agonism. At higher doses, t-MeHA levels gradually returned to basal levels, a profile probably resulting from agonism. After acute oral administration, betahistine increased t-MeHA levels with an ED50 of 2 mg/kg, a rightward shift likely due to almost complete first-pass metabolism. In each case, the maximal effect of betahistine was lower than that of ciproxifan, indicating partial inverse agonism. After an oral 8-day treatment, the only effective dose of betahistine was of 30 mg/kg, indicating that a tolerance had developed. These data strongly suggest that therapeutic effects of betahistine result from an enhancement of histamine neuron activity induced by inverse agonism at H3 autoreceptors.