To investigate the incidence and risk factors of ventilator-associated pneumonia (VAP) in neonatal intensive care unit (NICU), and to provide evidence for the prevention and control of VAP. A total of 1 872 neonates, who were admitted into NICU of Hunan Provincial Maternal and Child Health Hospital and subjected to mechanical ventilation from October 2016 to June 2018, were enrolled in the study. The neonates who met the diagnostic criteria of VAP were selected as the case group, and those who were treated with ventilator for 48 hours at the same time were regarded as the control group. Multivariate logistic regression model was used to analyze the related factors of VAP. Of the 1 872 neonates who underwent the mechanical ventilation, the VAP occurred in 160 cases with the incidence rate of 8.5% (160 cases). The 227 specimens were collected. Gram-positive bacteria (=116, 51.1%) were the main pathogens. The main pathogens were , , . By Chi-square test, birth weight, birth age, Apgar score, duration of ventilator, and whether newborn mothers with pregnancy hypertension were influencing factors. The result of logistic regression analysis showed that compared with no pregnancy included hypertension, the first aid measure at birth was initial resuscitation, and the MV time ≤ 5 days, the risk factors of ventilator-associated pneumonia in neonates included: their mothers with hypertensive disorders complicating pregnancy, using of tracheal intubation and ventilator time more than 5 days. The incidence of VAP in neonates receiving continuous MV therapy in neonatal intensive care unit is higher. Gram-positive bacteria are the main pathogens. VAP in neonates is related to whether newborn mothers with pregnancy hypertension, MV duration and tracheal intubation.