Intracellular reactive oxygen species (ROS) play an important role in cancer stem cell (CSC) function. Hesperetin (Hst) is a flavonoid that has been shown to affect cellular ROS level. The goal of this study was to investigate the effect of Hst on the level of ROS in gastric CSCs (GCSCs). MTT assay was used to evaluate cell survival. Cellular ROS level was measured using 2′,7′-dichlorofluorescin diacetate by flow cytometry. A significant decrease in the survival of GCSCs treated with Hst was observed. Hst decreased ROS level to about 47% and 40% at concentrations of 200 and 400 mM, respectively. It can be concluded that Hst reduces the survival and ROS level of GCSCs and may be influences the function of these cells such as proliferation and differentiation as well as response to chemotherapy by affecting cellular ROS level.