We previously demonstrated that the Runx3 transcription factor is expressed in the hypothalami, pituitaries, and ovaries of mice, and that Runx3 knockout (Runx3−/−) mice are anovulatory and their uteri are atrophic. Runx3 mRNA expression was detected in the granulosa cells of ovarian follicles, and in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). In the present study, we examined the effects of Runx3 knockout on the gene expression of enzymes associated with steroidogenesis. We found decreased Cyp11a1 mRNA expression in Runx3−/− mouse ovaries compared with that in wild-type (wt) mouse ovaries at the age of 8 weeks. In situ hybridization analysis showed that the percentages of Cyp11a1 mRNA-expressing theca cells in follicles of Runx3−/− mice were decreased compared with those of wt mice. In accord with the alterations in Runx3−/− mouse ovaries, Kiss1 mRNA levels in ARC were increased, whereas mRNA levels of kisspeptin in AVPV were decreased, and gonadotropin-releasing hormone in the preoptic area and follicle-stimulating hormone β subunit gene were increased in Runx3−/− mice. Following an ovarian transplantation experiment between Runx3−/− mice and wt mice, corpora lutea were observed when ovaries from Runx3−/− mice were transplanted into wt mice, but not when those from wt mice were transplanted into Runx3−/− mice, suggesting that Runx3 in the hypothalamo-pituitary system may drive gonadotropin release to induce ovulation in the ovary. These findings indicate that Runx3 plays a crucial role in the hypothalamo-pituitary-gonadal axis.