Objective: This study aims to investigate the relationship between serum 25-hydroxyvitamin D [25(OH)D] levels and pain severity in women with surgically confirmed endometriosis, according to disease phenotypes (Ovarian Endometrioma [OE] and Deep Infiltrating Endometriosis [DIE]). Methods: This retrospective cohort study included 427 women with a histopathologically confirmed diagnosis of endometriosis. Patients were divided into two groups: OE (n = 231) and DIE (n = 196). Serum 25(OH)D levels were categorized as deficient (< 20 ng/mL), insufficient (20–30 ng/mL), and sufficient (≥ 30 ng/mL). The severity of dysmenorrhea, chronic pelvic pain, and dyspareunia was measured using the Visual Analog Scale (VAS). Confounding factors such as seasonal variability, age, and BMI were controlled for using multivariate analyses. Results: A significant negative correlation was found between serum 25(OH)D levels and all pain parameters (p < 0.001). Median VAS scores were significantly higher in the group with vitamin D deficiency compared to the group with sufficient levels [7.0 (6.0–8.0) vs 4.0 (3.0–5.0), p < 0.001]. In a multivariate linear regression analysis, low 25(OH)D levels were identified as an independent predictor of pain intensity (β = -0.28, p < 0.001). The most striking finding was a significant interaction between vitamin D levels and disease phenotype (Interaction p = 0.004); this indicated that the exacerbating effect of low vitamin D on pain was much more pronounced in patients with the DIE phenotype compared to the OE group. A threshold value of 15.4 ng/mL demonstrated 74% sensitivity in predicting severe pain (VAS ≥ 7). Conclusion: Low serum vitamin D levels are directly associated with the severity of pain related to endometriosis. The dramatic strengthening of this association, particularly in the DIE phenotype, suggests that vitamin D may act as a critical modulator in neuro-inflammatory mechanisms within deep infiltrating lesions.