Recently, increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus (IAV) infections. Notably, some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans, posing a threat to public health. Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32 (ANP32) is the most important one among them. However, role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown. In the present study, a total of 10 feline ANP32 (feANP32) splice variants (including 5 feANP32A, 3 feANP32B, and 2 feANP32E) were obtained from domestic cats by RT-PCR. Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants, but all feANP32 proteins were primarily localized to cell nucleus. Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated. The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains, though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains. In addition, the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro. Collectively, our study systematically investigated the support activity of feANP32 on IAV, providing a clue for further exploring the mechanism of susceptibility of cats to IAV.