Objective To investigate the influence of pinealectomy（Px）and melatonin (MLT) supplementation on CD4SUP>+/SUP>CD25SUP>+/SUP>Treg cells development and Foxp3 expression in rat thymus. Methods One hundred and twenty clean-grade male SD rats were divided into five groups: normal control group, sham pinealectomized group, pinealectomized group, Px +7.5 mg/(kgI>&#/I>8226;d) MLT group, Px +15 mg/(kgI>&#/I>8226;d) MLT group, and the thymus were taken out at the 4th week and the 8th week; Flow cytometric analysis was used to analyse the number of double positive cells in the thymus and peripheral blood; Semiquantitative RT-PCR and immunohistochemistry were applied to analyse Foxp3 expression of rat thymus. Results There was no difference of the number of CD4SUP>+/SUP>CD25SUP>+/SUP> Treg cells in rat thymus between Px and normal/sham group, and the number increased dependently on time and dose after MLT supplementation. In rat peripheral blood the double positive cells significantly increased in models of Px at the 4th week, and then backed to normal level after MLT supplementation. The results showed no significant changes in all groups at the 8th weeek; At the 4th weeks, Foxp3 expression increased in the thymus of Px rats compared to nomal/sham group, which returned to normal level after MLT supplementation. On the other hand, Foxp3 expression showed no significant difference in all groups at the 8th weeek.Conclusion Px made no difference between the development of CD4+CD25+Treg cells in rat thymus, but resulted in significant increase of thymic output and Foxp3 expression. All the effects disappeared at the 8th week. MLT supplementation could reverse the abnormity.