Sp100 is a constitutive protein of nuclear domain 10 (ND10) and is ubiquitous in mammal cells. It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. Using a yeast mating assay, we found that Sp100 can interact with HIV-1 integrase (HIV-1 IN). This interaction was verified by co-immunoprecipitation, and intracellular imaging revealed that Sp100 and HIV-1 IN partially colocalized. Furthermore, mutant variants assay indicated that the C-terminal 300～480 residues of Sp100 and the catalytic domain of HIV-1 IN were responsible for this interaction. Knocking down endogenous Sp100 with Sp100-specific siRNA increased HIV-1 IN-mediated integration. Conversely, overexpression of Sp100 by transient transfection decreased HIV-1 IN-mediated integration. This is the first time that Sp100 has been found to interact with HIV-1 IN and inhibit lentiviral vector integration. It reveals a new function of Sp100 as a HIV-1 IN-interacting protein and expands knowledge of the cellular defensive response to viral infection.