The present study was performed to determine the behavioral effects of cholecalciferol (Vitamin D3) hormone treatment at different doses as an adjunctive therapy alone or in a combination with low dose of 17β-estradiol on depression-like behavior of female rats after long-term absence of estrogen. The aim of the study was to examine the effects of chronic cholecalciferol administration (1.0, 2.5 or 5.0 mg/kg/day, SC once daily, for 14 days) on depression-like behavior following long-term ovariectomy (12 weeks) of the adult (3 months old) female rats of Wistar line. Cholecalciferol was administered to the ovariectomized (OVX) rats and OVX rats treated with low dose of 17β-estradiol (17β-E2, 0.5 μg/rat, SC once daily, for 14 days) after long-term ovariectomy. Depression-like behavior was assessed in the forced swimming test (FST), locomotor and grooming activities were assessed in the open field test (OFT). Using biochemical studies were evaluated estradiol and 25-hydroxyvitamin D3 levels in the blood serum of OVX rats treated with cholecalciferol alone and cholecalciferol plus 17β-E2. Chronic administration of cholecalciferol (5.0 mg/kg/day, SC) into the intact females significantly reduced depression-like behavior in the FST (p<0.05). The treatment with cholecalciferol (1.0 mg/kg/day, SC) in the OVX rats after long-term absence of estrogens induced antidepressant-like effect (p<0.05) in the FST. Moreover, cholecalciferol in this dose plus 17β-E2 more markedly exhibited antidepressant-like effect in the OVX rats after longterm ovariectomy (p<0.05). Simultaneously, treatment with cholecalciferol (1.0 mg/kg/day, SC) in the OVX rats after longterm absence of estrogens produced elevated estradiol and 25-OH-VD3 levels for the OVX rats as compared to the OVX females. The combined application of cholecalciferol (2.5 and 5.0 mg/kg/day, SC) and 17β-E2 produced antidepressantlike effect that was similar to the antidepressant-like effect of 17β-E2. Our results indicate that cholecalciferol at dose of 5.0 mg/kg/day induced antidepressant-like effect only in intact rats subjected FST. Following long-term ovariectomy in the adult female rats, cholecalciferol at dose of 1.0 mg/kg/day administered alone resulted in decrease of depression-like behavior in the FST. Moreover, cholecalciferol at dose of 1.0 mg/kg/day in a combination with 17β-E2 at a low dose induced synergic antidepressant-like effect in the FST.