Leucovorin is one of the several chemically active reduced derivatives of folic acid. It is used as an antidote to folic acid antagonist. Lecovorin calcium inj U.S.P. contains calcium folinate which belongs to a group of vitamins, used as an antidote to the harmful effects of Methotrexate (MTX) therapy. In this investigation, the modulatory effects exerted by the Leucovorin against MTX induced genotoxicity in mice by using Chromosomal Aberrations (CA) and sperm morphology assays were tested. Three different doses of Leucovorin were selected and tested for their modulatory activity on the genotoxicity exerted by 20 mg/kg of MTX. The results of the present study indicate a significant decrease in the frequency of CA and sperm head abnormalities suggesting that the Leucovorin modulates the MTX induced genotoxicity in a dose dependent manner and it reveals its antitoxic nature and suggests using along with chemotherapeutic agents in cancer chemotherapy. These findings provide the future directions for the research on design and development of possible modulatory drugs containing Leucovorin.