<p>Aphthous stomatitis frequently affects oral mucosa, though its exact cause remains uncertain. Given vitamin D’s role in immune regulation and mucosal integrity, it may contribute to ulcer development. To assess the relationship between serum vitamin D levels and the occurrence, classification, and anatomical distribution of aphthous ulcers. This case-control study involved 74 patients diagnosed with aphthous stomatitis and 74 age- and sex-matched healthy controls. Data collected included demographic details, serum 25-hydroxyvitamin D levels, ulcer classification, and site of involvement. Vitamin D status was categorized as deficient (&lt; 20 ng/mL), insufficient (20–29 ng/mL), or sufficient (≥ 30 ng/mL). Statistical analysis was performed using the Chi-square test. Over half of the patients (54.1%) were aged 18–30 years, with no significant differences in age or sex between cases and controls. Vitamin D deficiency was significantly higher in patients (64.9%) than controls (24.3%) (<i>p</i> &lt; 0.001). Minor ulcers were the predominant type (82.4%), with deficiency significantly linked to both minor (<i>p</i> = 0.02) and major ulcers (<i>p</i> = 0.04). The labial mucosa was frequently involved site regardless of vitamin D levels, with no significant association between vitamin D status and ulcer location (<i>p</i> = 0.78). No significant differences by gender were observed in ulcer type (<i>p</i> = 0.79) or site (<i>p</i> = 0.92). Vitamin D deficiency is significantly associated with occurrence and severity of aphthous stomatitis, though it does not affect ulcer site or gender distribution. Assessing vitamin D status may aid in the management of these ulcers.</p>