Objective：To investigate the effects of the short-term estrogen replacement therapy（ERT） on the white matter and the myelin sheath ultrastructure of the myelinated fibers in the white matter of ovariectomized middle-aged rats in order to provide a sci－entific basis for delaying the process of brain aging and combating demyelination diseases with estrogen replacement therapy in the future. Methods：Middle-aged female sprague-dawley rats of 10-12 month（40 rats） were bilaterally ovariectomized and the serum e－strogen levels were examined one month after the surgery. The female Sprague-Dawley rats（19 rats） with successful castration were ran－domly divided into experimental group（n=10） and control group（n=9）. Experimental rats were subcutaneously injected 17β-estradiol and control rats were subcutaneously injected placebo for 4 weeks. Then，the spatial reference memory capacities of two group rats were tested with Morris water maze. The white matter and the myeli－nated nerve fibers in the white matter of two group rats were quantitatively investigated with the transmission electron mi－croscope technique and the stereological techniques. Results：The spatial reference memory capacity of the experimental rats was significantly enhanced compared with that of the control rats（F=9.645，P=0.006）. The total volume of the myelin sheaths and the difference between outer perimeter and inner perimeter of myelin sheaths in the myelinated fibers of the white matter in experimental rats were significantly increased compared with those of control rats（（32.94±5.97） mm3，（25.93±2.83） mm3，t=2.391，P=0.040；（1.16±0.10） ?滋m，（1.03±0.09） ?滋m，t=2.325，P=0.045）. The correlation analyses suggested that the total myelin sheath vol－ume of the myelinated fibers in the white matter was significantly and negatively correlated with the escape latency on the first day of Morris water maze（r=-0.744，P=0.009）. Conclusions：Estrogen replacement had positive protective effects on the spatial reference memory capacity and the myelin sheaths of the myelinated fibers in the white matter of the middle-aged ovariectomized rats.