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Supplemental digital content is available in the text.This systematic review was conducted to determine the prevalence of gram-negative bacteria in ventilator-associated pneumonia (VAP) as a potentially lethal and serious infection among mechanically ventilated neonates in neonatal intensive care units (NICUs). We searched PubMed, SCOPUS, EMBASE, and the ISI Web of Science databases as well as Google Scholar search engine up to November 20, 2017, with no restriction on language. We included studies reporting prevalence of gram-negative bacterial infection of neonatal VAP in NICUs. Risk of bias assessment using the tool developed by Hoy et al (J Clin Epidemiol. 2012; 65:934–939), and data extraction were conducted by 2 independent reviewers. A random-effects meta-analysis was used for combining the data. Of 11,072 screened references, 9 relevant studies were included in this meta-analysis. The overall pooled prevalence rate of gram-negative bacterial infections of neonates who were on mechanical ventilation for more than 48 hours was 72.0% (95% confidence interval, 0.60–0.83). Among the isolated gram-negative bacteria, Klebsiellaspecies had the highest detection rate, with 41.0% overall pooled prevalence. When the results were merged, the VAP rate was found to be 3/1000 ventilator-days. Male sex, prolonged hospitalization stay, and cesarean delivery were statistically significant risk factors for VAP incidence in NICUs. Prevalence of gram-negative bacteria in VAP in NICUs is high, and it is mainly caused by Klebsiellaspecies and could be affected by several factors. Further studies should be conducted on the drug resistance pattern of these bacteria. In addition, effective mechanisms for infection control should be considered to prevent these bacterial infections in VAP in NICUs.