Objective: To assess the safety and prebiotic effects of lactulose in preterm infants. Study design: This was a prospective, double-blinded, placebo-controlled, single-center study in 23- to 34-weeks premature infants. The study group received 1% lactulose, and control infants received 1% dextrose in all feeds (human milk or formula). Results: Twenty-eight infants participated (15 lactulose, 13 placebo). Small doses of lactulose appeared to be safe and did not cause diarrhea. Premature infants on lactulose had more Lactobacilli-positive stool cultures that appeared earlier with larger number of colonies. The lactulose group tended to have less intolerance to enteral feedings, to reach full oral feeds earlier, and to be discharged home earlier. They also tended to have fewer episodes of late-onset sepsis, lower Bell stage necrotizing enterocolitis, and their nutritional laboratory indices were better, especially calcium and total protein. Conclusions: This pilot study supports the safety of supplementing preterm infants' feeds with low doses of lactulose. It also demonstrated trends that may suggest positive prebiotic effects.