Influenza D virus (IDV) and influenza A virus (IAV) both use mRNA splicing to generate matrix (M1) and ion channel (M2) proteins, but via opposing strategies. IAV encodes M1 using unspliced mRNA1 and expresses M2 from spliced mRNA2. Conversely, IDV uses a spliced mRNA to encode M1, while the unspliced mRNA encodes a polyprotein that is thought to be post-translationally cleaved to produce DM2. Here, we compared IDV and IAV to elucidate how the distinct splicing strategies are regulated. Using minireplicon systems to recreate M segment splicing, we found that while the spliced IAV M segment is the minority species when expressed either by cellular RNA polymerase II or by the viral polymerase, the spliced IDV mRNA is the major product. However, for both viruses, removal of the NS segment increased splicing. We further demonstrated that the polypyrimidine (pY) tract upstream of the splice acceptor (SA) site is an essential element to regulate the splicing ratio for both IAV and IDV. In IAV, the pY tract consists of a purine-rich region preceded by pyrimidine stretch, maintaining relatively weak splicing. The IDV pY tract is a short pyrimidine stretch located immediately upstream of the SA. Purine substitution or insertion in the IDV pY tract markedly reduced splicing efficiency. Thus both viruses maintain the desired ratio of matrix to ion channel mRNA through fine tuning the strength of the cis-acting pY sequence and through evolving trans-acting anti-splicing functions in the NS segment.