Objectives: Colistin is a vital antibiotic used in multidrug-resistant infections. Its most important side effect is nephrotoxicity. Colistin is a weak acid. This study aims to evaluate whether urine alkalinization is protective in the nephrotoxicity of colistin. Methods: Twenty-eight male Sprague-Dawley rats were divided into groups. Group I (n = 4) was injected with intramuscular distilled water twice a day for 7 days. Group II (n = 8) was injected with 750,000 IU/kg/day colistin for 7 days. Group III (n = 8) was injected with the same dose of colistin after their urinary pH was >= 7 through the addition of bicarbonate in their drinking water. Group IV (n = 8) was injected with the same dose of colistin after their urine density fell below 1010 through the addition of NaCl molds in their food and 12.6 mg/L NaCl in their drinking water. Results: According to tubular degenerations (scored 0-5), group I scored 0, group II scored 4.25, group III scored 2, and group IV scored 1.5. In groups III and IV, protection was achieved (p = 0.001). The bicarbonate group was not superior to the NaCl group (p = 0.789). In transmission electron microscopy, group III had more microvilli integrity and autophagic vacuoles compared to group IV. Group IV had mitochondrial swelling and cristae lysis. A lower urine density was related to lower tubular scores (p = 0.001). Conclusions: Colistin was highly nephrotoxic without protection. Light microscopy findings revealed that urinary alkalinization and NaCl hydration were similarly protective. Urine alkalinization further prevents ultrastructural changes as revealed by electron microscopy.