Background: Neonatal sepsis has been a major cause of neonatal mortality and morbidity globally. Late onset sepsis is on the rise mostly due to better health care services and improved survival of premature neonates. Gram-negative sepsis has emerged as a major public health problem constituting significant morbidity and mortality. There is limited data on gram-negative late onset sepsis from the central part of India, therefore this study was conducted at a tertiary care center from rural part of India. Objectives: To determine the clinical profile and outcome among neonates with gram-negative late onset sepsis. Design: It is a retrospective analysis conducted among neonates with gram-negative late onset sepsis at a tertiary care center from central India. Methods: All neonates below 28 days of age suspected to have late onset sepsis were enrolled in the study. The data for the period of January 2019 to December 2021 was collected and analyzed using software SPSS version 29. The outcome variables studied were discharge (good outcome) and death (poor outcome). Results: In the present study, overall prevalence of gram-negative late onset sepsis was 4.8%. Respiratory distress (52.2%), seizure (18.9%), jaundice (15.6%), and lethargy (15.6%) were common clinical symptoms among neonates with sepsis. The most common organism isolated was Klebsiella spp. (36.7%) followed by Acinetobacter spp. (31.1%) and E. coli (17.8%). Low gestational age (n = 20 vs n = 7, P = .002) and low birth weight (n = 33 vs n = 4, P = .02) were associated with poor outcomes in neonates with gram negative LOS. The overall mortality rate was found to be 30% among neonates with gram negative sepsis. Conclusion: The prevalence of gram-negative sepsis was found to be 4.8%. Factors associated with poor outcome in gram-negative sepsis were low birth weight, and prematurity. Klebsiella spp. was found to be a common cause of gram-negative LOS, therefore, the empiric antibiotic policy must provide coverage against these micro-organisms.