Objective: The present study aimed to evaluate the effectiveness of N-Acetylcysteine (NAC), as an antioxidant, in preventing nephrotoxicity in patients receiving colistin. Methods: In a randomized controlled clinical trial, eligible participants receiving colistin were divided into two groups including drug ( n = 43) and control ( n = 39). In the drug group, 1200 mg of NAC was administered daily for 10 days concurrently with colistin. Patients in the control group received only colistin. The serum creatinine level (SCr), blood urea nitrogen (BUN), and creatinine clearance (CrCl) at baseline and every other day, and the number of cases with acute kidney injury (AKI) during the study were recorded. Before starting treatment and on day 5, the level of urinary neutrophil gelatinase-associated lipocalin (NGAL) was determined. Finally, the values were compared between the groups. Findings: There was a significant increase in SCr and BUN and a significant reduction in CrCl in both groups, but there was not any significant difference between the two groups at any time. Changes in the urine NGAL levels were not significantly different between the two groups. Even though the number of cases with AKI in the drug group (8 cases, 18.6%) was less than the control group (11 cases, 28.2%), the difference was not statistically significant ( P = 0.303). Conclusion: Simultaneous administration of NAC with a dose of 1200 mg daily does not have any effect in the prevention of colistin-induced nephrotoxicity.