The human proteins CDC5L (hCDC5) and PLRG1 are both highly conserved components of a multi-protein complex that is a subunit of the spliceosome. The proteins’ respective homologues in yeast are also associated with a sub-spliceosomal multi-protein complex that has been shown to be important for pre-mRNA splicing. Here we show that these two human proteins are associated in vivo and will interact directly in vitro. The interacting domains in both proteins have been identified. Our results indicate that the carboxyl terminal region of CDC5L and the WD40 domain of PLRG1 are essential for direct interaction between both proteins. By using a bacterially expressed mutant protein, containing the PLRG1 interacting domain in CDC5L, we show that the CDC5L-PLRG1 interaction in HeLa nuclear extract can be disrupted causing pre-mRNA splicing to be inhibited. Thus, a direct interaction between the CDC5L protein and PLRG1 in the CDC5L complex is essential for pre-mRNA splicing progression.