Swine influenza virus (SIV) poses a continuous threat to global pig production and public health. Efficient SIV replication is highly dependent on host factors, making their identification and functional characterization important for the development of host-directed antiviral strategies. Here, we investigated the role of the host factor SPART in SIV infection using CRISPR/Cas9-mediated knockout cells in combination with viral entry assays, flow cytometry, confocal microscopy, and stimulated emission depletion (STED) super-resolution imaging. SPART deficiency markedly impaired the replication of multiple influenza A virus (IAV) subtypes. Further analyses revealed that SPART knockout inhibited viral attachment. Mechanistically, SPART deficiency altered the organization of sialic acid clusters on the cell membrane by remodeling intracellular F-actin via hyperactivation of the MEK/ERK signaling pathway, without affecting the expression of sialic acid receptors. Collectively, these findings identify SPART as a novel host factor that promotes IAV attachment by maintaining sialic acid cluster organization, providing new mechanistic insight into host-mediated regulation of influenza virus entry.