Sepin-1 is a small compound that inhibits enzymatic activity of Separase and growth of cancer cells. As part of the IND-enabling studies to develop Sepin-1 as a chemotherapeutic agent, herein we have profiled the toxicity of Sepin-1 in Sprague-Dawley rats in a good laboratory practice (GLP) setting. The maximum tolerated dose (MTD) of Sepin-1 in rats is 40 mg/kg in single dose study and 20 mg/kg in the study dosed for 7 consecutive days. The toxicity study consists of two parts-Main Study and Recovery Study. Sepin-1 with 0 (control), 5 (low dose), 10 (median dose), and 20 (high dose) mg/kg was administered by bolus intravenous injection to rats once daily for 28 consecutive days. The animals in the Main Study were euthanized on Day 29, whereas animals in the Recovery Study were allowed to recover for 28 days following the 28-day Sepin-1 dose before they were euthanized on Day 29 of the off-dose period. Although the effects of Sepin-1 at low and median doses are minimal, hematological analysis shows that high-dose Sepin-1 is associated with decrease of red blood cells and hemoglobin, and increase in the number of reticulocytes and platelets as well as mean corpuscular volume. Clinical chemistry indicates that Sepin-1 causes increase of total bilirubin and decrease of creatine kinase. Histopathology analysis indicates Sepin-1 results in minimal bone marrow erythroid hyperplasia, minimal to moderate splenic extramedullary hematopoiesis, minimal splenic lymphoid depletion, minimal to mild thymic lymphoid depletion, and minimal to mild mandibular lymph node lymphoid hyperplasia in male and female rats in the Main Study. Those abnormal changes are Sepin-1 dose-dependent and mostly reversible after a 28-day recovery period in animals from the Recovery Study. Based on our results, we conclude that Sepin-1 at pharmacologic doses (5-10 mg/kg) is well tolerable, with no significant rates of mortality or morbidity, and can further be developed as a potential new drug to treat Separase-overexpressed tumors.