The segmented nature and high mutability of the influenza virus RNA genome facilitate rapid mutation and reassortment, allowing the virus to breach host barriers and migrate between different species, potentially leading to unpredictable influenza outbreaks. With dogs emerging as new natural hosts for influenza virus, vigilant surveillance and scientific prevention strategies are imperative. Here, based on our previous isolation of 21 strains, which are reassortments of the canine influenza virus (CIV) H3N2 (KR/07) with gene segments from the influenza A pandemic (H1N1) 2009 virus strain (CA/09), the replication kinetics of these reassortants in immortalized mammalian respiratory epithelial cell lines from swine and ferret named hTERT-PBECs and hTERT-FBECs, alongside induced changes in cytokine expression, were investigated. Reverse genetics was utilized to generate the reassortment H3N2 canine influenza rKR/07-PB2/NP, which contains the PB2 and NP segments from CA/09. The viral titer of rKR/07-PB2/NP was significantly lower than those of the parental viruses KR/07 and CA/09. In addition, rKR/07-PB2/NP notably decreased expression levels of interleukin-1β (IL-1β) and interleukin-10 (IL-10) in both immortal cells, particularly in hTERT-PBECs. Our findings not only contribute to the understanding and exploring cross-species transmission mechanisms of influenza virus, but also provide new ideas for prevention and treatment of CIV.