Background. Previous epidemiological studies revealed that anterior cruciate ligament (ACL) injuries were more frequently seen in female athletes than in male athletes. To elucidate the pathogenetic roles of estrogen in ACL ruptures, the elasticity of ACL tissue was measured using a scanning acoustic microscope (SAM) in an estrogen-controlled animal model. Methods. A total of 40 ovariectomized Japanese white rabbits were randomly divided into four groups according to the administered dose of 17β-estradiol (groups L, M, H, and C). Injection of 17β-estradiol was performed 1, 2, 3, and 4 weeks after surgery, and doses in groups L, M, and H were 50, 100, and 500 μg/kg, respectively. Group C received no estradiol. Only groups L, M, and C were used for current analyses because their mean serum estrogen levels were within the physiological range (groups C, L, M, and H: 37, 50, 60, and 231 pg/ml, respectively). Five weeks after ovariectomy, the lateral portion of the ligament was harvested. Specimens were fixed with 10% neutralized formalin and embedded in paraffin. Then, 10 μm thick sections were cut perpendicular to the ligament fibers for routine histological staining and measurement with SAM. Results. The mean tissue sound speeds of groups C, L, and M were 1727 ± 32, 1683 ± 53, and 1665 ± 63 m/s, respectively. Group M presented significantly lower tissue sound speed than group C (P = 0.021). Furthermore, a negative correlation was found between the mean serum estrogen level and mean tissue sound speed of the ACL among all animals in groups C, L, and M (r = -0.47, P = 0.016). Conclusion. The results of the present study indicated that estrogen altered the tissue elasticity of rabbit ACL. Estrogen may constitute one of the pathogenetic factors in ACL rupture in female athletes.