Ro 25-6981 is a highly potent and selective blocker of N-methyl-d-aspartate receptors that has been shown to possess both rapid and sustained antidepressant activity. In the present study, we report the biopharmaceutical characterization of Ro 25-6981 by evaluating gastrointestinal stability, transepithelial permeability, stability in human liver microsomes, and in silico metabolic prediction. Moreover, in vivo efficacy of Ro 25-6981 after oral administration was evaluated in animal models of depression. When mixed with 5 different simulated gastrointestinal fluids, no loss of parent compound was observed after 6 h, indicating compound stability in the gastrointestinal environment. At the tested concentrations, Ro 25-6981 was shown to have transepithelial permeability with apparent permeability (Papp) values comparable to highly permeable drugs. Ro 25-6981 was metabolized within 30 min in human liver microsomes, and the metabolic prediction data showed glucuronidation and sulfation as potential metabolic pathways. The in vivo efficacy data suggested that Ro 25-6981, when administered orally at 30 mg/kg, exhibits antidepressant-like activity following oral administration with efficacy comparable to traditional antidepressants that is both dose- and time-dependent. Overall, due to optimal gastrointestinal stability, oral permeability, and oral efficacy, Ro 25-6981 can be a potential therapeutic option for the treatment of depression.