Background: Some members of the genus Hypericum have been shown to demonstrate antidepressant-like effects. In Colombia, approximately 54 species of Hypericum have been reported, and only a few have been investigated chemical and pharmacologically. Objective: The aim of this study was to evaluate the antidepressant-like effects of a methanol extract, as well as ethyl acetate and butanol fractions, obtained from aerial parts of Hypericum juniperinum K. Materials and Methods: Behavioral and locomotor activities were evaluated in the open field test (OFT). Antidepressant-like activity was measured in the forced swimming test (FST) in male Swiss albino mice. Preliminary phytochemical screening as well as a high-performance liquid chromatography (HPLC) profile of the active fractions of H. juniperinum was performed. Results: Treatment with the methanol extract at 500 mg/kg and the ethyl acetate and butanol fractions at 150 and 300 mg/kg resulted in a decrease in the immobility times in FST. In the OFT, none of the treatments presented altered behavior or locomotor activity of mice. Preliminary phytochemical screening identified terpenes/steroids, flavonoids, phenol derivatives, tannins, and saponins. In the HPLC analysis of the fractions, rutin, quercitrin, and quercetin were identified with the help of coinjection of standards. Conclusion: This is the first report of the antidepressant-like activity of extracts and fractions obtained from H. juniperinum in the FST model of depression. Flavonoids may be responsible for the antidepressant-like action of H. juniperinum.