Etimicin sulfate, an ethylization derivative of gentamicin, is a new soluble wide-spectrum synthetic aminoglycoside drug. It has wide antibacterial spectrum with high effect and less cross resistance as compared to other aminoglycosides. In order to further explore its safety and tolerance, we have conducted a subactute toxicity study on swiss albino mice. Results from the present study have elucidated that treatment of etimicin sulfate exerts no significant signs of toxicity at any dose level used in the study. Physiological as well as hematological parameters were unaltered throughout the study. Biochemical examination and histopathology of all organs confirmed no significant alteration at any dose levels. The result of this study has suggested there was no obvious toxicity observed with the treatment of etimicin sulphate. It was found to be a safe alternative for various severe infections.