Subfertility affects about 10-15% of Malaysians, characterized by difficulty conceiving after 12 months of unprotected intercourse. Emerging research suggests that vitamin D deficiency, influenced by dietary intake and sleep patterns, may contribute to fertility. This study examines the relationship between chrononutrition patterns, sleep quality and vitamin D deficiency in subfertile patients. A cross-sectional study was conducted at two hospitals in Selangor, Malaysia, involving 140 subfertile individuals who fit the inclusion and exclusion criteria. Data were collected through self-administered questionnaires, including food frequency questionnaires (FFQ), the Chrononutrition Profile Questionnaire (CPQ), and the Pittsburgh Sleep Quality Index (PSQI). Serum vitamin D levels were measured through 25-hydroxyvitamin D blood test. Statistical analyses were performed using SPSS version 29. Results revealed a significant difference in mean serum vitamin D levels between male and female participants (p < 0.001), with 76.7% of the females being vitamin D deficient. Most parameters were not significant in males compared to females. Notably, a negative correlation was found between vitamin D levels and both energy and fat intake in females (p < 0.05). Female gender was identified as a significant determinant of vitamin D deficiency (OR 5.186, p < 0.001), while poor evening eating habits were strongly associated with deficiency (AOR 10.553, p < 0.05). These findings highlight the importance of gender and dietary patterns in vitamin D deficiency among subfertile patients. Hence, there is a need for targeted nutritional and lifestyle interventions.