The present study focused on the molecular characterization and toxicity analysis of the Bacillus thuringiensis Berliner (Bt) isolate T121 against Henosepilachna vigintioctopunctata (F.) (Coleoptera: Coccinellidae). Colonies of T121 growing on solid media were circular, with creamy white hues and raised centers. Scanning electron microscopy revealed the presence of cuboidal and spherical crystals in liquid media. Polymerase chain reaction screening confirmed the presence of binary toxin vip1 and vip2 in addition to cry3Aa gene. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed four prominent bands at 74, 65, 48, and 31 kDa. In toxicity assays, T121 exhibited a median lethal concentration of 4.675 µg/ml against H. vigintioctopunctata. These findings demonstrate that the Bt isolate T121 offers promising prospects for development as a component of a sustainable pest management strategy against H. vigintioctopunctata and perhaps other coleopteran pests.