This study was designed to determine the antiproliferative activity of three extracts (alcoholic, hydro-aqueous and aqueous) and their fractions from the root part of Calotropis procera using human oral (KB) and central nervous system (SNB-78) cancer cell lines as a model system. KB and SNB-78 cells were cultured in the presence of extracts and fractions at various concentrations (10, 30 and100 μg/ml) for 48 h, and the percentage of cell viability was evaluated by the sulforhodamine-B (SRB) assay. Our result indicates that out of the three extracts of C. procera (root), alcoholic extract had shown greater potential for growth inhibition followed by hydro-aqueous extract at three different concentration of 10 μg/ml, 30 μg/ml and 100 μg/ml in a dose-dependent manner, whereas aqueous extract was found to be least active against both oral and CNS human cancer lines. On evaluation of the fractions prepared from alcoholic and hydro-aqueous extracts, it was observed that chloroform fraction from alcoholic extract was antiproliferative for oral (KB) cancer cell line and n-butanol fraction from alcoholic extract was antiproliferative for CNS cancer cell line than remaining fractions at three different concentration of 10 μg/ml, 30 μg/ml, 100 μg/ml in a dose-dependent manner. Thus, our result indicates that the root part of C. procera possess in vitro cytotoxicity against oral and CNS human cancer cell lines. Further investigations are required to obtain the clinically important lead molecules for the drug development.