Animal models occupy an irreplaceable position for investigating the pathogenesis and treatment of autoimmune diseases. Although different laboratories have adopted adenovirus expressing human TSHR A-subunit (Ad- TSHR289) to induce a Graves' disease (GD) animal model, intervention manners are substantially different. The aim of this study is to investigate the effect of the time and frequency of administration of Ad-TSHR289 on the development of a Graves' disease animal model. Forty-five Female BALB/c mice were injected with Ad-TSHR289 or control Ad-lacz twice (5w) or thrice (10w) at three-weekly intervals. Blood and thyroid glands were obtained 2 wk or 4 wk after injection. Our data showed that all of the mice injected with Ad-TSHR289 had significantly higher TRAb compared with the control group. Fifty-six percent (9/16) of 5w- and 75% (9/12) of 10w-model group mice became hyperthyroid as judged by serum T4 levels. The consistency between serum T4 levels and histological changes of thyroid was 50% in 5w- and 100% in 10w-model group. The 10 week intervention is thus better than 5 week to induce a GD animal model for higher incidence and consistency, suggesting that intervention time and frequency are two important factors affecting the development of a GD animal model induced by Ad-TSHR289. © 2012 American Scientific Publishers. All rights reserved.