Background and Objective Omeprazole is a proton pump inhibitor that is used in acid suppression therapy in infants. Infants cannot swallow the oral tablets or capsules. Since, infants require a non-standard dose of omeprazole, the granules or tablets are often crushed or suspended in water or sodium bicarbonate, which may destroy the enteric coating. In this study we explore the efcacy and pharmacokinetics of rectally administered omeprazole in infants with gastroesophageal refux disease (GERD) due to esophageal atresia (EA) or congenital diaphragmatic hernia (CDH) and compare these with orally administered omeprazole. Methods Infants (6–12 weeks postnatal and bodyweight >3 kg) with EA or CDH and GERD were randomized to receive a single dose of 1 mg/kg omeprazole rectally or orally. The primary outcome was the percentage of infants for whom omeprazole was efective according to predefned criteria for 24-h intraesophageal pH. Secondary outcomes were the percentages of time that gastric pH was <3 or <4, as well as the pharmacokinetic parameters. Results Seventeen infants, 4 with EA and 13 with CDH, were included. The proportion of infants for whom omeprazole was efective was 56% (5 of 9 infants) after rectal administration and 50% (4 of 8 infants) after oral administration. The total refux time in minutes and percentages and the number of refux episodes of pH<4 decreased statistically signifcantly after both rectal and oral omeprazole administration. Rectal and oral administration of omeprazole resulted in similar serum exposure. Conclusions A single rectal omeprazole dose (1 mg/kg) results in consistent increases in intraesophageal and gastric pH in infants with EA- or CDH-related GERD, similar to an oral dose. Considering the challenges with existing oral formulations, rectal omeprazole presents as an innovative, promising alternative for infants with pathological GERD.