AimsWe studied whether hyperbaric oxygen (HBO2) treatment, which is known to increase production of nitric oxide (NO) in the brain, might also produce an NO-dependent anxiolytic-like behavioral response. Main methodsMale NIH Swiss mice (20–25g) were subjected to a 60-min HBO2 treatment at different absolute atmospheres, and anxiety was assessed using the light/dark exploration test at different time intervals following the cessation of HBO2 treatment. To ascertain the underlying mechanism of action, other groups of mice were pretreated with the NO synthase inhibitor NG-monomethyl-l-arginine acetate, the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), the soluble guanylyl cyclase-inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the benzodiazepine antagonist flumazenil to determine their influence on the HBO2-induced anxiolytic-like effect. Key findingsA 60-min HBO2 treatment at 3.0 absolute atmospheres increased the time spent by mice in the light compartment that lasted up to 90min following the end of HBO2 treatment. This anxiolytic effect of HBO2 was significantly reduced by pretreatment with L-NMMA, carboxy-PTIO, ODQ and flumazenil. SignificanceBased on these findings, we conclude that a 60-min HBO2 treatment is capable of inducing an anxiolytic effect that possibly involves NO, cyclic GMP and the benzodiazepine binding site.