Considering the role of oxidative stress as a molecular mechanism underlying the deleterious side effects of gentamicin, applying antioxidants is likely to have ameliorative effects against GNT-induced tissue damages. This work was aimed to investigate the protective effect of betaine or trimethylglycine upon gentamicin-induced toxicity in mice. Thirty male mice were randomly divided into five groups: group 1 (control; i.p. injection of isotonic saline), group 2 received gentamicin (GNT; 80 mg/kg, i.p.) for 10 days, group 3 received GNT (80 mg/kg, i.p.) for 10 days and betaine (2% in the diet) for 18 days starting 8 days before gentamicin injection, group 4 received GNT (80 mg/kg, i.p.) for 10 days and betaine (2% in the diet) during gentamicin prescription, and group 5 just received betaine (2% in the diet) for 10 days. Gentamicin administration caused a notable increase in creatinine and urea levels compared to controls (p < 0.05). Betaine administration in groups 3 and 4 decreased creatinine values relative to the second group to the amounts that had no significant difference in comparison with the control group. Additionally, a remarkable increase in renal and plasma contents of malondialdehyde (as a lipid peroxidation indicator) and renal glutathione peroxidase (which breakdown hydrogen peroxides and hydroperoxides to harmless molecules) was found upon gentamicin injection which both were attenuated in the betaine-treated groups. Also, the histopathologic studies revealed acute tubular necrosis with hyperemia and hemorrhage triggered by gentamicin. Betaine noticeably attenuated the GNT-induced histopathological lesions of the kidneys. The present study indicates that betaine can attenuate gentamicin-induced nephrotoxicity, which might be related to betaine’s antioxidant potential.