OBJECTIVE To explore the influences of orthodontic tooth movement on estrous cycle and estrogen in female rats. METHODS Two hundred female rats were divided into control group, one-time activation group, four-time activation group and sham-operated group. Each group was divided again into 4 sub-groups according to the different stage of the estrous cycle. The force-loading groups received repeated intermittent force. Serum estrogen was measured and the change of estrous cycle was recorded. RESULTS There were significant variations in the estrogen level among the groups which received mechanical force during the same stage of the estrous cycle (P < 0.01) and among the subgroups within each group (P < 0.05). These experimental treatments affected the estrous cycle of the rats in the groups received force in metestrus and diestrus. CONCLUSIONS Estrus of rats was the appropriate time for the orthodontic force.