Levamisole is an antihelminthic drug originally developed for veterinary medicine, and it is also used in human medicine due to its immunomodulatory effects. Recently, it has been outstanding for its reported benefits in the treatment of COVID-19. Furthermore, levamisole inhibits monoamine oxidase-A (MAO-A), which plays an essential role in the pathophysiology of anxiety. This study aimed to investigate the effects of levamisole on anxiety-like behaviors assessed in adult male rats by the light-dark box test (LDB) and the open field test (OFT). The animals were divided into two groups (n=10 per group): vehicle and levamisole. The vehicle group was administered distilled water, while the levamisole group received levamisole (2 mg/kg) by oral gavage daily for 4 weeks. After the last levamisole treatment, LDB and OFT experiments were performed. Levamisole significantly enhanced the latency to enter the dark compartment (p=0.025). Moreover, the number of transitions decreased with levamisole treatment (p=0.021). Nevertheless, levamisole did not alter the time spent in the light and dark compartments (p=0.363 for both). In the OFT, the number of entries into the central area (p=0.782), the total number of crossed lines (p=0.250), the number of fecal pellets (p=0.336), the total number of rearings (p=0.367), and the duration of grooming (p=0.187) remained unchanged with levamisole administration. However, levamisole increased the time spent in the central area, while it decreased the time spent in the peripheral area (p=0.049 for both). Chronic levamisole administration may stimulate anxiolytic-like effects in adult male rats, without affecting locomotor and exploratory activities.