People who drink water contaminated with arsenic for a long time develop neuritis, cerebellar symptoms, and deficits in memory and intellectual function. Arsenic induces oxidative stress and promotes apoptosis through multiple signalling pathways in nerve cells. Neuroglobin (Ngb), as a key mediator, is considered to be protective against oxidative stress. In this study, we aimed to study the effects of Ngb knockdown in arsenite-treated rat neurons on levels of apoptosis markers and reactive oxygen species and serum Ngb levels of subjects from arsenic-endemic regions in China. We discovered that arsenic-induced apoptosis and reactive oxygen species production were enhanced in Ngb-knocked-down rat neurons. Silencing of Ngb aggravated the arsenic-induced decrease in the rate of Bcl-2/Bax and the levels of Bcl-2 protein following arsenite treatment. The results also showed that serum Ngb levels were independently negatively correlated with arsenic concentration in drinking water. Furthermore, the serum Ngb levels of four groups (245 individuals) according to different degree exposure to arsenic were 815.18 ± 89.52, 1247.97 ± 117.18, 774.79 ± 91.55, and 482.72 ± 49.30 pg/mL, respectively. Taken together, it can be deduced that Ngb has protective effects against arsenic-induced apoptosis by eliminating reactive oxygen species.