PRPF40A and PRPF40B are two splicing factors which play an important role in alternative splicing. Despite the high similarity of their protein sequences, PRPF40A and PRPF40B are not redundant in function. This study focused on the functional analysis of PRPF40A in comparison to its paralogue, PRPF40B. To this purpose, PRPF40A and PRPF40B sequences were both divided into three fragments. These fragments were swapped separately to form six PRPF40 Hybrids (AAB, ABA, BAA, ABB, BAB, BBA) which were subsequently overexpressed in human myeloid cell. By comparing the PRPF40 Hybrids, we discovered that the WW domains and the intrinsically disordered region are the determinant regions of PRPF40A and PRPF40B. The splicing regulatory pattern of PRPF40A and PRPF40B was also revealed. PRPF40A included the cassette exons adjacent to the introns with high GC content, and excluded the cassette exons adjacent to the introns with low GC content. PRPF40B has the opposite splicing regulation pattern.