Intracellular reactive oxygen species (ROS) accumulation during culture process compromise cord blood-derived hematopoietic stem/progenitor cells (CB-HSPCs) proliferation potential dramatically. In this work, the effects of hyperoside (Hyp) on CD34(+) cells were investigated by the analysis of cell expansion and secondary expansion potential, with the objective to optimize the culture conditions for ex vivo expansion of HSPCs. The results showed that the expansion of total cells treated with 1 mu M Hyp reached 54.9 +/- 9.6 folds, which was significantly higher than that of control cells (42.0 +/- 8.1 folds, P < 0.05), however, the percentages of CD34(+) cells and CD34(+) CD38(-) cells were similar in two cultures, leading to significantly higher expansion of CD34(+) cells and CD34(+) CD38(-) cells in Hyp-treated cultures (P < 0.05). Importantly, it was found that CD34(+) cells cultured with Hyp possessed better secondary expansion ability and colony forming potential, which meant better self-renewal potential. Furthermore, the addition of 1 mu M Hyp reduced the ROS level in total cells, CD34(+) cells and CD34(+) CD38(-) cells, and protected cells from apoptosis (P < 0.05). Together, Hyp is beneficial for the ex vivo expansion of functional HSPCs, which can be possibly related to its capability of downregulating the intracellular level of ROS.