Vitamin D3 (VD3) supplements increase pregnancy rates. In addition to its effect on fertilized eggs, the involvement of VD3 in endometrial decidualization, which is essential for embryo implantation, has been suggested; however, the detailed mechanisms involved remain unclear. The present study examined the effects of VD3 on endometrial decidualization and embryo implantation using the human endometrial stromal cell line, KC02‑44D, and the choriocarcinoma cell line, BeWO. The effects of VD3 were examined using reverse transcription‑quantitative PCR (RT‑qPCR) and changes in the invasive capacity of BeWO cells were examined using invasion assay. The results revealed that VD3 further elevated the levels of heart and neural crest derivatives expressed 2 (HAND2), whose protein is a master regulator that is elevated during decidualization, and VD3 suppressed elevated prolactin (PRL) in decidualized KC02‑44D cells, as shown by RT‑qPCR analysis. The addition of VD3 to decidualization reduced the invasive capacity of BeWO cells in the invasion assay. A manual search for the vitamin D receptor binding motif suggests that HAND2 may be directly controlled by VD3. Given that VD3 regulates PRL, VD3 supplementation would be appropriate to avoid the endometrial secretory phase and provide VD3 during the endometrial proliferative phase, with the expectation of an effect on fertilized eggs.

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