Irritable bowel syndrome (IBS) and vitamin D deficiency are common among children in Latin America. Previous studies show that Bifidobacterium longum35624^TM improves IBS symptoms in adults. This real-world, single-arm, open-label study conducted in Chile investigated the effects of B. longum 35624 (1 × 10⁹ colony-forming units, 12 weeks) on gastrointestinal symptoms (adapted IBS severity scoring system [IBS-SSS]; adapted Questionnaire on Pediatric Gastrointestinal Symptoms [QPGS], and Bristol Stool Form Scale) in 64 children and adolescents (8–18 years) and explored the relationship with baseline vitamin D status. Improvements in all IBS-SSS domains and composite score were observed at week 6 and 12 (p < 0.0007 versus baseline), with 98.3% of participants experiencing numerical improvements in ≥3 domains. Clinically meaningful improvement was seen in 96.6% of participants. The distribution of IBS-SSS severity categories shifted from moderate/severe at baseline to mild/remission (p < 0.0001). Improvements were not maintained during the two-week washout. Low baseline serum vitamin D levels did not correlate to IBS severity or probiotic response. QPGS significantly decreased from baseline to week 6 (p = 0.0005) and 12 (p = 0.02). B. longum 35624 may improve IBS symptoms in children and adolescents, even those with vitamin D deficiency. A confirmatory randomized controlled trial and further exploration of probiotic response and vitamin D status are needed.