Oxidative stress and mitochondrial dysfunction are related to disease pathogenesis. Oligodeoxynucleotide containing CpG motifs (CpG ODN) demonstrate possibilities for immunotherapy applications. The aim of the present work is to explore the underlying mechanism of the cytoprotective function of CpG ODN by employing the oxidative stress modulation in immune cells. We used the imaging flow cytometry to demonstrate that tert-butyl hydroperoxide (t-BHP) induces mitochondrial-mediated apoptosis and ROS production in RAW264.7 cells. After pretreatment with CpG ODN, the percentage of apoptotic cells and ROS production was both markedly reduced. The decrease in mitochondrial membrane potential (MMP) induced by t-BHP was partially reversed by CpG ODN. The t-BHP induced upregulation of the expression of apoptosis-related proteins (cleaved-caspase 3, cleaved-caspase 9, cleaved-PARP, and bax) was notably decreased in the presence of CpG ODN. Furthermore, we found that CpG ODN enhanced phosphorylation of ERK1/2 and Akt to inhibit ROS production. In conclusion, the protective effect of CpG ODN in mitigation of t-BHP-induced apoptosis is dependent on the reduction of ROS.