Introduction: Urinary intestinal fatty acid binding protein (i-FABP), a marker of intestinal mucosal cell damage, has recently been proposed as a clinically useful measure in the early detection of necrotizing enterocolitis. However, there are no data on urinary i-FABP in meconium obstruction of prematurity (MOP). This study aimed to evaluate urinary i-FABP in MOP patient as a marker for early detection. Methods: This investigation was a prospective cohort study conducted in the neonatal intensive care unit of Seoul National University Children’s Hospital between October 2012 and September 2015 including preterm infants weighed 1,250g at birth. We collected the initial urine samples at 12, 24, and 48 hours after birth (±6 hours), and further urine samples were collected every 48 hours till the amount of feed reached to 40 ml/kg/day. The urinary i-FABP levels were compared between the control group and the MOP group. Results: A total of 91 infants with a median gestational age (GA) of 28.1 weeks and a median birth weight of 940 g were analyzed. During the study period, 15 patients were diagnosed with MOP. Urinary i-FABP levels were significantly associated with GA and birth weight. There was a significant correlation between initial urinary i-FABP levels and the time of full enteral feeding achievement after adjusting GA. Urinary i-FABP/creatinine levels were significantly increased at 48 hours after birth in MOP patients (5.65 ± 8.34 pg/nmol vs. 8.95 ± 7.05 pg/nmol, p = 0.025). Conclusion: Urinary i-FABP levels were inversely associated with GA and birth weight, and also significantly increased among preterm infants with MOP compared with the control group at the time of 48 hours after birth. Measurements of initial urinary i-FABP levels may be helpful for early detecting of MOP and predicting of feeding intolerance.