Background Preterm infants with long-term parenteral nutrition (PN) therapy are at risk for cholestasis associated with total parenteral nutrition (PNAC). This study examined the safety and efficacy of ursodeoxycholic acid (UDCA) in preventing PNAC in preterm infants. Our research aimed to investigate the prophylactic effect of preventive oral UDCA on PNAC in preterm infants. Methods We compared oral administration of UDCA prophylaxis with no prophylaxis in a randomized, open-label, proof-of-concept trial in preterm neonates with PN therapy. The low-birth-weight preterm infants (< 1800 g) who were registered to the neonatal intensive care unit (NICU) within 24 hours after birth were randomized. The main endpoint was the weekly values of direct bilirubin (DB) of neonates during the NICU stay. Results Eventually, a total of 102 preterm neonates from January 2021 to July 2021 were enrolled in this prospective study (42 in the UDCA group and 60 in the control group). Notably, the peak serum level of DB [13.0 (12-16) vs. 15.2 (12.5-19.6) mu mol/L, P < 0.05)] was significantly lower in the UDCA group than that in the control group without prevention. The peak serum level of total bilirubin (101.1 +/- 34 vs. 116.5 +/- 28.7 mu mol/L, P < 0.05) was also significantly lower in the UDCA group than in the control group. Furthermore, the proportion of patients who suffered from neonatal cholestasis (0.0% vs. 11.7%, P < 0.05) in the UDCA group was significantly lower. Conclusion UDCA prophylaxis is beneficial in preventing PNAC in NICU infants receiving prolonged PN.