The aim of the current study was to examine the effects of chronic cholecalciferol administration (1.0, 2.5 or 5.0 mg/kg/day, s.c., once daily, for 14 days) on the anxiety-like and depression-like behaviors following long-term ovariectomy (12 weeks) in female rats. Cholecalciferol was administered to the ovariectomized (OVX) rats and OVX rats treated with 17β-estradiol after long-term absence of estrogen (17β-E2, 0.5 µg/rat, s.c., once daily, for 14 days). Anxiety-like behavior was assessed in the elevated plus maze (EPM), depression-like behavior was assessed in the forced swimming test (FST), locomotor and grooming activities were assessed in the open field test (OFT). The treatment with cholecalciferol (1.0 mg/kg/day, s.c.) in the OVX rats after long-term absence of estrogens induced antidepressant-like effect (p<0.05). Moreover, cholecalciferol in this dose plus 17β-E2 more markedly exhibited antidepressant-like effect in the OVX rats after long-term ovariectomy (p<0.05). The OVX rats treated with cholecalciferol at doses of 1.0 mg/kg and 2.5 mg/kg demonstrated a decrease of anxiety-like behavior in the EPM. The combination of cholecalciferol at doses of 1.0 and 2.5 mg/kg with a low dose of 17β-E2 more effectively decreases anxiety-like behavior in the OVX rats after long-term estrogen deﬁciency than17β-E2 alone. This work promotes more effective creation of novel therapeutic targets and strategies for affective-related disorders treatment in female subjects with long-term estrogen deﬁciency.