3,3'-Diindolylmethane (DIM), a novel poison targeting Leishmania donovani topoisomerase I (LdTOP1LS), induces programmed cell death in Leishmania parasites. The development of resistant parasites by adaptation with increasing concentrations of DIM generates random mutations in LdTOP1LS. Single-nucleotide mutations result in the amino acid substitutions F270L and K430N in the large subunit and N184S in the small subunit of the enzyme. DIM failed to inhibit the catalytic activity of the recombinant mutant enzyme (LdTOP1DRLS). Transfection studies of the mutant genes showed that the mutated topoisomerase I confers DIM resistance on wild-type Leishmania parasites. Site-directed mutagenesis studies revealed that a substantial level of resistance is conferred by the F270L mutation alone; however, all three mutations (F270L, K430N, and N184S) together are required to reach a higher-resistance phenotype. DIM fails to stabilize the topoisomerase I-DNA covalent complexes in the F270 mutant. Moreover, DIM cannot interfere with the religation step in the catalytic cycle of the recombinant F270L mutant enzyme. Taken together, these findings identify novel mutations in topoisomerase I that hinder its interaction with DNA, thereby modulating enzyme catalysis and conferring resistance to DIM. These studies advance our understanding of the mechanism of cell poisoning by DIM and suggest a specific modification of the drug that may improve its efficacy.