Vitamin D plays a critical role in reproductive health, with emerging evidence suggesting it may influence fertility outcomes in patients undergoing Assisted Reproductive Techniques (ART). This study investigates the effects of serum vitamin D levels on ART outcomes, evaluating ovarian response, embryo quality, and pregnancy rates. We conducted a comparative analysis of ART patients stratified by serum 25(OH)D levels: deficient (<20 ng/mL), insufficient (20–30 ng/mL), and sufficient (>30 ng/mL). A total sample size of 300 was calculated using EPI software to ensure statistical power. Results demonstrated a statistically significant association between vitamin D sufficiency and enhanced ovarian response, with higher oocyte retrieval (mean ± SD: 12.5 ± 3.6 in sufficient vs. 9.1 ± 2.8 in deficient groups, p < 0.01) and implantation rates (p < 0.05). Sufficient vitamin D levels also correlated with improved embryo quality and live birth rates, emphasizing the potential benefit of vitamin D optimization in ART patients. These findings highlight the importance of considering vitamin D as a modifiable factor in fertility protocols.