Macrophages contribute to HIV‐1 pathogenesis by forming a viral reservoir that serve as a viral source for the infection of CD4 T cells. The relationship between HIV‐1 latent infection and superinfection in macrophages has not been well studied. Using susceptible U1 cells chronically infected with HIV‐1, we studied the effects of HIV superinfection on latency and differences in superinfection with HIV‐1 and HIV‐2 in macrophages. We found that HIV‐1 (MN) superinfection displayed increased HIV‐1 replication in a time‐dependent manner; while cells infected with HIV‐2 (Rod) initially showed increased HIV‐1 replication, followed by a decrease in HIV‐1 RNA production. HIV‐1 superinfection upregulated/activated NF‐ĸB, NFAT, AP‐1, SP‐1, and MAPK Erk through expression/activation of molecules, CD4, CD3, TCRβ, Zap‐70, PLCγ1, and PKCΘ in T cell receptor‐related signaling pathways; while HIV‐2 superinfection initially increased expression/activation of these molecules followed by decreased protein expression/activation. HIV superinfection initially downregulated HDAC1 and upregulated acetyl‐histone H3 and histone H3 (K4), while HIV‐2 superinfection demonstrated an increase in HDAC1 and a decrease in acetyl‐histone H3 and histone H3 (K4) relative to HIV‐1 superinfection. U1 cells superinfected with HIV‐1 or HIV‐2 showed differential expression of proteins, IL‐2, PARP‐1, YB‐1, and LysRS. These findings indicate that superinfection with HIV‐1 or HIV‐2 has different effects on reactivation of HIV‐1 replication. HIV‐1 superinfection with high load of viral replication may result in high levels of cytotoxicity relative to HIV‐2 superinfection. Cells infected with HIV‐2 showed lower level of HIV‐1 replication, suggesting that co‐infection with HIV‐2 may result in slower progression toward AIDS. J. Cell. Physiol. 232: 1746–1753, 2017. © 2016 Wiley Periodicals, Inc.