Asparagus racemosus Linn. (AR) is used worldwide as a medicinal plant. In the present study, the anxiolytic activity of standardized methanolic extract of root of AR (MAR) was evaluated in open-field test (OFT), hole-board, and elevated plus maze (EPM) tests. Rats received oral pretreatment of MAR in the doses of 50, 100, and 200 mg/kg daily for 7 days and then were evaluated for the anxiolytic activity in different animal models. Both MAR (100 and 200 mg/kg) and diazepam (1 mg/kg, p.o.) increased the grooming behavior, number of central squares crossed, and time spent in the central area during OFT. Further, MAR (100 and 200 mg/kg) increased the head-dip and head-dip/sniffing behavior, and decreased sniffing activity in hole-board test. Furthermore, MAR (100 and 200 mg/kg) increased the percentage entries and time spent to open arm in EPM test paradigm. The anxiolytic activity in the experimental models was similar to that of diazepam. MAR (100 and 200 mg/kg) enhanced the level of amygdalar serotonin and norepinephrine. It also increased the expression of 5-HT2A receptors in the amygdala. In another set of experiment, flumazenil attenuated the anxiolytic effect of minimum effective dose of MAR (100 mg/kg) in OFT, hole-board, and EPM tests, indicating GABAA-mediated mechanism. Moreover, the anxiolytic dose of MAR did not show sedative-like effect in OFT and EPM tests compared to diazepam (6 mg/kg, p.o.). Thus, the anxiolytic response of MAR may involve GABA and serotonergic mechanisms. These preclinical data show that AR can be a potential agent for treatment of anxiety disorders.