Compared with mammalian ANP32A, most avian-coded ANP32A contains a 33 amino acids insertion (ch-ANP32A-33) or a 29 amino acids insertion (ch-ANP32A-29), which can rescue the mammalian-restricted avian influenza virus polymerase activity, with ch-ANP32A-33 exhibiting a more potent phenotype. The alternative splicing of 3′ splice sites (SSs) of chicken ANP32A intron 4 generates full-length ch-ANP32A-33 and truncated ch-ANP32A-29. In this study, we found a splicing regulatory cis-element that affected the alternative splicing of 3′ SSs by block-scanning mutagenesis. RNA affinity purification and mass spectrometry showed that the SRSF10 bound to the splicing cis-element and the binding was further identified and confirmed by RIP experiment. Overexpression of SRSF10 changed the ratio of the two chicken ANP32A transcripts with the increased ch-ANP32A-29 and the decreased ch-ANP32A-33. The knockdown of both of the ch-ANP32A-33 and ch-ANP32A-29 was harmful to avian influenza virus polymerase activity in DF-1 cells, but the restoration and increasement of only ch-ANP32A-29 could not completely rescue the activity of avian influenza virus polymerase. Overexpression of SRSF10 negatively affected the polymerase activity and replication of avian influenza virus, and the expression of ch-ANP32A-33 could partially recover the decrease of polymerase activity of avian influenza virus. By contrast, SRSF10 had weak inhibition on the polymerase activity of mammalian adapted influenza virus and had no effect on the replication of mammalian adapted influenza virus. Taken together, we demonstrated that SRSF10 acts as a negative regulator in polymerase activity and replication of avian influenza virus by binding to the splicing cis-element to regulate the alternative splicing of chicken ANP32A intron 4 for the reduced ch-ANP32A-33 and increased ch-ANP32A-29.Copyright © 2020. Published by Elsevier B.V.