Abstract: This study aimed to elucidate the effects of alfalfa flavonoids on reproductive performance, reproductive organs, hormone level, and gene expression in the hypothalamus-pituitary-ovarian gonadal axis in pregnant animals. Three doses of alfalfa flavonoids (120, 400 and 1200 mg/kg) and two control groups (diethylstilbestrol 0.5 mg/kg, blank control group) were used. The middle and high doses of alfalfa flavonoids significantly (P<0.05) increased the level of estradiol (E2) (P<0.05), while the low dose of alfalfa flavonoids highly significantly (P<0.01) increased the level of E2. There was no evident difference of progesterone level between the groups. The number of live fetus significantly (P<0.05) increased in three doses of alfalfa flavonoids, the weight of live fetus was highly significantly (P<0.01) increased with the high dose of alfalfa flavonoids. There was no evident difference in the number of luteum although there was a declining trend. Moreover, compared with control group, diethylstilbestrol, reduced the number of luteum. Compared with the control group, the three doses of alfalfa flavonoids significantly (P<0.05) increased the litter size, and the newborn litter weight increase was highly significant (P<0.01). Expressions of GnRH in the hypothalamus and FSH in the pituitary were highly significantly (P<0.01) reduced by alfalfa flavonoids and diethylstilbestrol. The expression of FSHR in the ovaries was reduced by the high dose of alfalfa flavonoids, but the expression of LHR was increased. The expressions of other genes showed no significant difference compared with the control group. In conclusion, the reproductive performance of rats was improved, because the alfalfa flavonoids influenced the hypothalamus-pituitary-ovarian gonadal axis.