Genistein potentiates the anti-cancer effects of gemcitabine in human osteosarcoma via the downregulation of Akt and nuclear factor-κB pathway. Genistein, a nontoxic flavonoid compound, has potent antitumor activity in various cancer cells. In the present study, we investigated whether genistein could be employed as a novel strategy to enhance the anti-tumor activity of gemcitabine using human osteosarcoma MNNG/HOS tumor model. In vitro, by MTT, electron microscopy, immunobloting and qRT-PCR assay, we found that the combination treatment of genistein and gemcitabine resulted in stronger growth inhibition and apoptosis induction through the downregulation of NF-κB activity and Akt activation in osteosarcoma cells. Moreover, the synergetic effects were observed when genistein was replaced by PI3K/Akt-pathway inhibitor (LY-294002) or NF-κB inhibitor (BAY11-7082). In vivo, the combination therapy augmented tumor growth inhibition through the down-regulation of NF-κB activity and Akt activation in xenografts. Taken together, these results provide in vitro and in vivo evidence that genistein abrogates gemcitabine-induced activation of NF-κB and increases the chemosensitization of osteosarcoma to gemcitabine. Combination therapy appears as a rational and novel approach for osteosarcoma treatment.