Highly pathogenic avian influenza (HPAI)-H7N9 virus arising from low pathogenic avian influenza (LPAI)-H7N9 virus with polybasic amino acid substitutions in the haemagglutinin was detected in 2017. We compared the tropism, replication competence and cytokine induction of HPAI-H7N9, LPAI-H7N9 and HPAI-H5N1 in ex vivo human respiratory tract explants and in vitro culture of human alveolar epithelial cells (AECs) and pulmonary microvascular endothelial cells (HMVEC-L). Replication competence of HPAI- and LPAI-H7N9 were comparable in ex vivo cultures of bronchus and lung. HPAI-H7N9 predominantly infected AECs, while limited infection was observed in bronchus. The reduced tropism of HPAI-H7N9 in bronchial epithelium may explain the lack of human-to-human transmission despite a number of mammalian adaptation markers. Apical and basolateral release of virus was observed only in HPAI-H7N9 and H5N1 infected AECs regardless of infection route. HPAI-H7N9, but not LPAI-H7N9 efficiently replicated in HMVEC-L implying that endothelial tropism may involve in pathogenesis of HPAI-H7N9 disease.