The acid-sensing ion channels (ASICs) are present on the cellular membranes of virtually all mammalian central neurons, but their physiological role still remains to be discovered. Here, we described the effect of novel orthosteric ASIC antagonist, compound 5b, on rat’s hippocampal integral electrical signal exposed to the open field behavior test. The analysis of experimental rat’s EEGs revealed that 5b-administered animals demonstrate substantial decrease in theta rhythm frequency. Lowering the theta rhythm frequency is specific for anxiolytics - pharmacological agents (e.g. benzodiazepines, barbiturates, etc.), that are used in medicine for anxiety-related disorders treatment. Under 5b, the animal behavior was also significantly altered from that in control: in rats with suppressed ASIC channels, the locomotor function was reduced similarly to the effect of a large dose of anxiolytics. Using small-molecule orthosteric ASIC antagonist, we characterized for the first time the effect of pharmacological blockade of these channels on electrical activity of hippocampus and behavioral reactions of rats. Our data support the results of previous studies on the participation of ASIC channels in regulation of behavioral reactions and negative emotional states such as anxiety and depression.