Objective: To explore the risk factors of ventilator-associated pneumonia (VAP) in premature infants with respiratory distress syndrome (RDS) undergoing assisted ventilation. Methods: A total of 100 premature infants with RDS who received assisted ventilation in Quanzhou First Hospital from May 2020 to May 2022 were selected as observation targets. The incidence of VAP during assisted ventilation was counted. According to the occurrence of VAP, they were divided into VAP group and non-VAP group. The risk factors of VAP during assisted ventilation in premature infants with RDS were analyzed by univariate and multivariate logistic regression analysis. Results: There were 34 premature infants of VAP and 66 premature infants of non-VAP in 100 premature infants with RDS during assisted ventilation, and the incidence of VAP was 34% (34/100). Univariate analysis showed that the time of invasive mechanical ventilation treatment, the time of non-invasive ventilation treatment after extubation, the rate of neonatal asphyxia, the rate of neonatal rescue, and the number of repeated intubations in the VAP group were higher than those in the non-VAP group, the difference was statistically significant (P＜0.05). Multivariate Logistic regression analysis showed that the time of invasive mechanical ventilation treatment, the time of non-invasive ventilation treatment after extubation, neonatal asphyxia, neonatal rescue, and the number of repeated intubations >3 times were independent risk factors for VAP during assisted ventilation treatment in preterm infants with RDS (P＜0.05). Conclusion: There is a high possibility of VAP in premature infants with RDS during assisted ventilation due to factors such as mechanical ventilation treatment time, neonatal asphyxia and repeated intubation.