Objective Gender differences in immune cell types in peripheral blood and spleen were compared between aged and young Sprague-Dawley rats. Methods Twelve-month and 2-month-old rats were used. Rats in each age group were half male and half female. Classification and counts of peripheral blood cells, organ index, peripheral blood and spleen immune cell typing, and P16 expression in spleen T cells were determined and the result compared with respect to age and gender differences. Results The peripheral blood leucocyte count was decreased in the aged female rats, and there was a gender difference. The erythrocyte count was increased in both aged male rats and aged female rats, but there was a difference between them. The neutrophil percentage was increased and the lymphocyte percentage was decreased in the aged male and female rats. The eosinophil percentage was increased in the aged male and female rats. The monocyte percentage in the aged female rats was lower than that in the aged male rats. No significant changes in other peripheral blood indices were found. The thymus index of the aged female rats was decreased and was lower than that of the aged male rats. The spleen index of the aged male rats was increased and was higher than that of the aged female rats. Peripheral blood immunophenotyping indicated that the percentages of T helper cells, regulatory T cells and cytotoxic T cells were increased in the aged female rats. The percentage of B cells was decreased in the aged female rats. The percentage of natural killer cells was increased in the aged male rats. Splenic lymphocyte typing showed that numbers of CD3+ cells increased and CD45RA+ cells decreased in aged female rats. P16 expression in splenic T cells increased in all aged rats, and no gender difference was observed. Conclusions No gender differences were found in the various indices among young rats. Some indices showed a gender difference in aged rats, including peripheral blood leucocyte count, erythrocyte count, neutrophil percentage, monocyte percentage, thymus index, spleen index, and immune cell typing for regulatory T cells, cytotoxic T cells, natural killer cells, B cells and CD3-positive cells. These result indicate altered immune system homeostasis in aged rats, leading to gender differences in some indices in aged rats. The present result provided basic data for studying the impact of gender on aging-related diseases and animal models of aging.