The prevalence of patent ductus arteriosus (PDA) in preterm infants is high. There is little information about the therapeutic effect and safety of rectal acetaminophen in the treatment of PDA. We aimed to compare the therapeutic effect and safety of oral and rectal acetaminophen on PDA in preterm infants. This study was a single-blind randomized clinical trial using 40 preterm infants. The cases were hospitalized in the neonatal intensive care unit of Mohammad Kermanshahi and Imam Reza hospitals of Kermanshah. Subjects were randomly divided into 2 groups, the first group was treated with oral acetaminophen and the second group was treated with rectal acetaminophen. The presence of PDA and response to treatment was assessed based on pre- and post-treatment echocardiographic criteria. The likelihood of complications or prohibition of acetaminophen use was assessed with paraclinical tests before and after treatment. The neonates were in the age range of 30 to 35 weeks. Twenty-one cases (52.5%) were boys and 19 cases (47.5%) were girls. Two cases in the oral-acetaminophen group and 1 case in the rectal-acetaminophen group needed the second round of treatment. There was no difference between the success of treatment and the type of treatment. The study showed that there was no difference between PDA treatment of preterm infants with oral and rectal acetaminophen. Also, no side effects were observed in treatment with any of the treatments. Therefore, it could be suggested that in infants who are intolerant to oral acetaminophen, the rectal form can be used.