The global dissemination of H5 avian influenza viruses represents a significant threat to both human and animal health. In this study, we conducted a genome-wide siRNA library screening against the highly pathogenic H5N1 influenza virus, leading us to the identification of 457 cellular cofactors (441 proviral factors and 16 antiviral factors) involved in the virus replication cycle. Gene Ontology term enrichment analysis revealed that the candidate gene data sets were enriched in gene categories associated with mRNA splicing via spliceosome in the biological process, integral component of membrane in the cellular component, and protein binding in the molecular function. Reactome pathway analysis showed that the immune system (up to 63 genes) was the highest enriched pathway. Subsequent comparisons with four previous siRNA library screenings revealed that the overlapping rates of the involved pathways were 8.53%–62.61%, which were significantly higher than those of the common genes (1.85%–6.24%). Together, our genome-wide siRNA library screening unveiled a panorama of host cellular networks engaged in the regulation of highly pathogenic H5N1 influenza virus replication, which may provide potential targets and strategies for developing novel antiviral countermeasures.