Objective: Cancer is one of the most deadly types of disease and evasion from apoptosis and unstoppable cell proliferation are accepted as its distinctive features. Many studies have evaluated the cytotoxic effect of different Morus species but, there is no study about cytotoxic effect of Morus rubra. In this study we aimed to evaluate phenolic composition, antioxidant properties and cytotoxic effect of acidified dimethyl sulfoxide extract of M. rubra (AMRE). Method: Antioxidant properties, phenolic composition and cytotoxic effect of AMRE were determined using spectrophotometric methods, HPLC, and MTT assay, respectively. Then, mechanisms of cytotoxic effect of AMRE on human prostate (PC-3) and lung (A549) cancer cells were examined in regard to cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. Results: Total phenolic content and reducing power values were 11.9 mg gallic acid equivalents and 42.9 mg trolox equivalents per g sample, respectively. Ascorbic and gallic acid were detected in AMRE as major antioxidant compounds. We determined that AMRE increased cell cycle arrest at G(1) phase and exhibited apoptotic features via decreasing mitochondrial membrane potential in both prostate and lung cancer cells. Conclusion: These findings demonstrate that M. rubra extract can affect the behavior of human prostate and lung cancer cells in vitro conditions, and this effect now needs to be investigated in vivo.