Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors specifically involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins for activity on a model short 56-nucleotide intron-containing HNRNPH1 pre-mRNA. We identified a known alternative splicing regulator SPF45 (RBM17) as a general splicing factor that is essential to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells revealed that SPF45 is specifically required for the efficient splicing of many short introns. Our crosslinking and biochemical analyses demonstrate that SPF45 specifically replaces the U2AF heterodimer on the truncated poly-pyrimidine tracts in these short intron. To initiate splicing, the U2AF-homology motif (UHM) of the replaced SPF45 interacts with the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.