IntroductionVarious approaches are employed to expedite the passage of meconium in preterm infants within the neonatal intensive care unit (NICU), with glycerine enemas being the most frequently used. Due to the potential risk of high osmolality-induced harm to the intestinal mucosa, diluted glycerine enema solutions are commonly used in clinical practice. The challenge lies in the current lack of knowledge regarding the safest and most effective concentration of glycerine enema. This research aims to ascertain the safety of different concentrations of glycerine enema solution in preterm infants.Methods and analysisThis study protocol is for a single-centre, two-arm, parallel-group, double-blind and non-inferiority randomised controlled trial. Participants will be recruited from a NICU in a teriary class A hospital in China, and eligible infants will be randomly allocated to either the glycerine (mL): saline (mL) group in a 3:7 ratio or the 1:9 ratio group. The enema procedure will adhere to the standardised operational protocols. Primary outcomes encompass necrotising enterocolitis and rectal bleeding, while secondary outcomes encompass feeding parameters, meconium passage outcomes and splanchnic regional oxygen saturation. Analyses will compare the two trial arms based on an intention-to-treat allocation.Ethics and disseminationThis trial is approved by the ethics committee of the Medical Ethics Committee of West China Second University Hospital of Sichuan University. The results will be published in a peer-reviewed journal.Trial registration numberChiCTR2300079199.