Several studies have reported that vitamin D may modify human reproductive functions; however, the results are conflicting. We aimed to comprehensively evaluate serum vitamin D levels and examine the relationship between serum vitamin D levels and ovarian reserve markers, and immune markers of implantation, in reproductive-aged Japanese women with infertility.in reproductive-aged women with infertility. This cross-sectional, single-center study included reproductive-aged women who underwent preconception screening for fertility. Serum vitamin D levels and reproductive and immune markers were measured. Standard and advanced statistical techniques were used. We observed a statistically significant difference in the seasonal and monthly 25(OH) vitamin D levels; the 25(OH) vitamin D level during winter was the lowest among all seasons. However, there was no linear correlation between 25(OH) vitamin D levels and ovarian reserve markers, such as follicle-stimulating hormone and anti-Müllerian hormone, or the Th1/Th2 cell ratio, which is used as an implantation-related immunological marker. In this large-scale study, we evaluated the serum 25(OH) vitamin D concentration in reproductive-aged women with infertility in Japan; however, there was no association between reproductive function and vitamin D levels.