**Abstract**

The present study was performed to determine the behavioral effects of cholecalciferol treatment at different doses as an adjunctive therapy alone or in a combination with low dose of 17β-estradiol on anxiety-like behavior of the middle-aged (12–14 months) and old (16–18 months) female rats at 12 weeks after ovariectomy. Vitamin D3 supplementation individually (as cholecalciferol at doses of 1.0, 2.5 or 5.0 mg/kg/day, s.c.) or in co-administration with 17β-estradiol (17β-E2, 0.5 μg/ rat, s.c.) were given to the ovariectomized (OVX) rats of different age at 12 weeks after ovariectomy. Anxiety-related state was tested in the elevated plus maze (EPM) and light-dark test (LDT), as well behavioral reactivity was registered in the open field test (OFT). The results of the study indicated that vitamin D3 supplementation at doses of 1.0 or 5.0 mg/kg/day decreased manifestations of anxiety-like profile in the middle-aged or old OVX rats, respectively. Vitamin D3 (1.0 mg/kg/day) plus 17β-E2 resulted in more profound anxiolyticlike effects in the old OVX rats than effects of both drugs administered alone. Cholecalciferol (1.0 mg/kg/day, s.c.) in the old OVX rats produced elevated estradiol and 25-OH-VD3 levels for these rats as compared to the old OVX females treated with solvent.

**Keywords:** vitamin D3, anxiety, menopause, estradiol, aging, female rats
