Osteoblast-activating peptide (OBAP) is a novel protein affecting osteoblast proliferation and differentiation, but its ovarian expression is yet to be reported. Osteoporosis is a common disease, caused mainly by low estrogen levels in females. We investigated whether OBAP regulates estrogen synthesis and osteoporosis. Using immunohistochemical analyses, we studied the distribution of OBAP in different parts of the mouse ovary. We also attempted to clarify the correlation of OBAP with ovarian steroids and calcium-regulating factors in the same ovarian tissues, including aromatase (CYP19), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), estrogen receptor (ER), progesterone receptor (PR), receptor activator of nuclear factor-kappa B (RANK), calmodulin, calbindin, and calcium-sensing receptor. The ovarian interstitial endocrine cells (IC) showed the greatest localization of OBAP, followed by the mature corpus luteum and the oocytes of mature Graafian follicles (MGF), while there were strong negative correlations of OBAP with CYP19. Strong positive correlations with 3 beta-HSD (except MGF), RANK (except IC), and calmodulin (except MGF and IC) were demonstrated. OBAP also showed partially positive correlations with ER and PR in the corpus luteum and with IC and calbindin in the MGF. We conclude that OBAP might be related to estrogen synthesis and calcium homeostasis.