Pre-mRNA splicing is a critical step in gene expression for metazoans. Several viral proteins regulate the splicing of pre-mRNAs through complex interactions with the host cell RNA splicing machinery. Here, we focus on a novel function of HIV-1 Vpr, which selectively inhibits cellular and viral pre-mRNA splicing via interactions with components of the spliceosome complex. This review discusses our current knowledge of how RNA splicing regulation is accomplished by Vpr-host factor interactions.