In order to study the effects of dietary calcium on the expression of uncoupling protein 3(UCP3) in skeletal muscle of Wistar rat fed with high fat diet, 54 male Wistar rats were randomly divided into 6 groups: Group A was fed with basic diet(0.5% calcium), Group B was fed with high fat low calcium diet(0.08% calcium), Group C was fed with high fat normal calcium diet(0.5% calcium), Group D and E was fed with high fat high calcium diet(1% and 1.5% calcium respectively), Group F was fed with high fat high calcium diet (1.5% calcium) supplemented with vitamin D(600 IU%). Reverse transcription polymerase chain reaction (RT-PCR) was used to measure the level of UCP3 mRNA. The results showed that the body weight and fat content in group C were significantly higher than those in group A. However, the body weight gain in groups of D, E, and F was less than those in group C. The serum insulin in group C increased significantly compared with that in groups of A, D, E and F(P < 0.05), but there was no significant difference in blood glucose among all groups. Compared with group A, the level of serum leptin increased significantly in group C(P < 0.05) and increased slightly in groups D, E and F (P > 0.05). The level of serum FT3 in groups D, E and F increased significantly compared with that of group A, B and C(P < 0.05), but there was no significant difference in FT4 among all groups(P > 0.05). Compared with group A, the expression of UCP3 mRNA decreased in skeletal muscle in group C, while UCP3 mRNA increased in groups D, E and F compared with that of group C. In conclusion, dietary calcium may not only reduce the body weight gain induced by high fat diet, and improve hyperinsulinemia, but also may enhance the expression of UCP3 mRNA in skeletal muscle by increasing the serum level of leptin and FT3 which may play the role in the prevention of obesity.