Identifying the host factors that mediate avian influenza virus adaptation in mammals is important for monitoring zoonotic potential. Although viral polymerase adaptations are known to influence cross-species transmission, the engagement of specific host factors with divergent viral polymerases remains to be explored. We examined human DEAD-box RNA helicases (DDXs) as potential regulators of influenza polymerase activity. A screen of 16 DDXs identified DDX10 as a factor that selectively enhanced the polymerase activity and replication of human-adapted H1N1 viruses, including the 2009 pandemic strain, but not avian-origin H9N2 viruses. This differential activity was associated with DDX10 showing stronger interaction with human-origin PB1 (Cal04/H1N1) than with avian-origin PB1 (BJ16/H9N2). Strain specificity was mediated by two residues (336 and 364) within the PB1 catalytic domain: introducing H1N1-type residues (I336 and I364) into H9N2-PB1 conferred DDX10 responsiveness, while reciprocal mutations in H1N1-PB1 abolished it. This work defines DDX10 as a potential host factor that differentially supports influenza polymerase activity, revealing a specific molecular interface that contributes to the replication efficiency of distinct viral subtypes in human cells and providing insight into host-adaptive mechanisms.