Background <p>We investigated the applicability of human decellularized ovarian scaffold (HDOS) to create an artificial ovary for preserving germ cells and forming follicular-like structures in a mouse chemotherapy-induced premature ovarian failure (chemo-POF) model after seeding stromal cells and transplantation.</p> Methods <p>The present study was conducted in three steps. (1) Creation of a mouse POF model, (2) Production of an artificial ovary (AO), (3) Transplantation of AO to ovariectomized mice. For the first step, female NMRI mice received chemotherapy drugs to induce POF. For AO production, mouse ovarian cell suspension (OCS) from the POF model and the control group were isolated separately and seeded into human decellularized ovarian scaffolds (HDOSs). AO was transplanted to ovariectomized intact and POF mice, which were followed up for 2 months (AOII). After transplantation, histological assessments, hormonal evaluation (FSH/E2), and gene expression analysis were conducted (<i>Blimp1</i>).</p> Results <p>A follicle-like structure formed two months after the transplantation. No significant increase was observed in the FSH level in both the Intact/AOII and POF/AOII groups. The gene expression of <i>Blimp1</i> showed a significant increase in the Intact/AOII group compared to the other groups.</p> Conclusions <p>The bioengineered ovary is capable of forming a follicle-like structure in the POF model after cell seeding and transplantation. We also demonstrated the efficacy of these extracellular matrices (ECMs) in preserving germ cells.</p>