There is growing interest in the proposed antineoplastic activity of Nerium oleander components. Earlier we have developed an oleander callus culture. The objectives of this work were to reveal whether the cultured tissue is able to generate secondarymetabolites representative of the parent plant under in vitro conditions and whether these products retain biological activities of the source. The callus tissue produces oleandrin, oleandrigenin and odoroside, the main oleander secondary metabolites. The levels of cytotoxicity of callus and plant leaf extracts for human cell lines in vitro are comparable. Callus extract expresses species-specific cytotoxicity characteristic of the parent plant. The both extracts tested are toxic for human (normal embryo fibroblasts, Jurkat,K-562, L-41,KB cell lines) andmonkey (COS-7 cell line) cells, while rat PC-12 cell line has low sensitivity to the extract of leaves and is fully resistant to callus extract. The extracts were shown to induce apoptosis. The anti-genotoxic and anti-mutagenic activity of polysaccharide fractions of oleander leaves and callus culture was revealed and can be considered to be novel findings. Thus, oleander callus culture retains the main activities of the plant. This culture can be used as a biotechnological source of biologically/pharmacologically active compounds of oleander.