Macrophages are divided into two types: M1-and M2-type macrophages. Both types of macrophages serve important roles during the process of inflammation. M1-type macrophages release various pro-inflammatory cytokines, such as IL-1, IFN-γ and other inflammatory mediators, such as nitric oxide, glutamate and reactive oxygen species to generate inflammation. In contrast, M2-type macrophages counteract the pro-inflammatory M1 conditions and promote tissue repair by secreting anti-inflammatory cytokines, such as IL-10. In spinal cord injury (SCI), an imbalance in M1/M2 macrophages leads to irreversible tissue destruction. Thus, it is crucial to increase the number of M2-type macrophages and promote M2 polarization of macrophages in SCI. Accordingly, in this study an in vitro co-culture system was established to investigate the effect of neural stem cells (NSCs) on macrophages. The results of the present study demonstrated that NSCs induced M2 polarization and suppressed M1 polarization of macrophages in an interleukin (IL)-4-dependent manner. Furthermore, the nuclear factor (NF)-κB/p65 signaling pathway was involved in the M1 polarization of macrophages and NSCs suppressed the activation of the NF-κB/p65 pathway in an IL-4-dependent manner to induce M2 macrophage polarization. These findings provide more insight into SCI and help to identify novel treatment strategies.