Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator‐induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro‐surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)‐8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP‐A, SP‐B, SP‐C, SP‐D), CK‐8, KL‐6, αENaC, and aquaporin (AQP)‐3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP‐C, cytokines, and intercellular adhesion molecule‐1 was vigorous in response to stimulation with tumor necrosis factor‐α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells.