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Aim: For many years, colistin has not been considered a first-line treatment due to its toxic side effects. However, its use has recently been reevaluated as a last resort in the treatment of neonatal infections caused by multidrug-resistant (MDR) gram-negative bacteria (GNB). Accordingly, in this study we aimed to contribute to the literature by investigating the efficacy and nephrological effects of colistin use in neonates. Materials and Methods: The retrospective study was conducted in the neonatal intensive care unit of a tertiary university hospital between January 2015 and February 2019 and included 30 patients who received intravenous treatment with colistin for culture-proven hospital-acquired GNB infections. We analyzed the serum sodium, potassium, phosphate, calcium and magnesium levels, urea, creatinine, aspartate aminotransferase and alanine aminotransferase values, and urine outputs measured on the 1st, 3rd, and 10th days of treatment. Results: All patients were treated with colistin for at least 10 days (mean 16.07±3.22 days) and the treatment dose was 5 mg/kg per day. We observed a statistically significant difference between the 1st- and 10th-day urea, creatinine, calcium, magnesium, and urine output values (p˂0.05). Similarly, when we compared the measurements recorded on the 1st and 10th days of treatment, we found that the magnesium and calcium levels were significantly decreased (p=0.008 and p=0.038, respectively) while the urea, creatinine, and urine output values were significantly increased (p=0.027, p=0.022, p=0.001, respectively). Discussion and Conclusion: Colistin is an effective agent in the treatment of MDR-GNB infections in neonates. Neonates should be closely monitored for nephrotoxicity during treatment with colistin. The efficacy and safety of neonatal treatment with colistin should be investigated with further, larger-sample studies.