Aim: To identify the parasporin-producing, indigenous Bacillus thuringiensis strains that specifically targets human cancer cells in Madurai, Tamil Nadu, South India. Methods and Results: Alkali-solubilized inclusion proteins from the 82 nonclonal indigenous isolates of B. thuringiensis were analysed for their cytotoxicity against two human cancer cell lines, U-937 (human histiocytic lymphoma) and HCT-250 (adherent human colon cancer cells). Activated inclusion protein from one of the isolates, B. thuringiensis LDC-391, was found to be highly cytotoxic to HCT-250 and moderately toxic to U-937, but nontoxic to normal lymphocytes. This strain did not show any insecticidal activity against the lepidopteran and dipteran larvae tested, as well as it was nonhaemolytic on human erythrocytes. The Western-blotting analysis showed that the putative 180 kDa cytotoxic protein from the isolate B. thuringiensis LDC-391 cross-reacted with the reference antisera of 81-kDa parasporin-1. Conclusions: Our observations imply that B. thuringiensis LDC-391 is different from the already reported parasporin producers, as it is showing variation in the target specificity. Significance and Impact of the Study: Characterizing these proteins can pave the way to alleviate problems associated with neoplastic transformation and cancer progression.