In this study, we aimed to clarify the protective effect of umbilical cord-derived mesenchymal stem cell conditioned medium (UC-MSC-CM) on neuronal oxidative injury and its potential mechanism. Neuronal oxidative damage was mimicked by H2O2 treatment of the HT22 cell line. Immunofluorescence staining was used to analyze the expression of cleaved Caspase3 and TRPM2. Cytotropic factors secreted by UC-MSCs were analyzed by enzyme-linked immunosorbent assay. Whole-cell patch clamping was used to detect TRPM2-like currents. Western blotting was utilized to analyze the protein levels of SOD, p-ERK1/2, p-JNK1/2/3 and cleaved-Caspase-9. The number of cleaved-Caspase-3-positive cells and protein expression of Caspase-9 induced by H2O2 treatment were decreased by UC-MSC-CM treatment. Furthermore, SOD protein expression was increased in the MSC-CM group compared with that in the H2O2 group. The H2O2-induced TRPM2-like currents in HT22 cells were attenuated by MSC-CM treatment. In addition, H2O2 treatment downregulated the expression of p-JNK protein in HT22 cells, and this the downward trend was reversed by incubation with MSC-CM. Thus, we showed that UC-MSC-CM protects neurons against oxidative injury, possibly by inhibiting activation of TRPM2 and the JNK signaling pathway.