Natural killer (NK) cells are known as innate immune lymphocytes that respond rapidly when challenged by pathogens but little is known about adaptive immune features including memory related to NK cells from human beings. In the present study, we demonstrate for the first time that human NK cells expressing the memory‐associated marker CD45RO were persistent in pleural fluid cells (PFCs) from tuberculous patients. CD45RO⁺ NK cells produced significantly more interferon‐γ and were more cytotoxic compared with CD45RO⁻ NK cells from PFCs when stimulated with interleukin‐12 (IL‐12). Consistently, IL‐12 enhanced the expression of granzyme B, CD69, CD25, NKG2D, IL‐12 receptors β1 and β2 on CD45RO⁺ NK cells from PFCs. Our experiments contribute to a better understanding of the NK cells from PFCs and indicate that human CD45RO⁺ NK cells from PFCs expressing a ‘memory‐like’ phenotype may have an important role in defending against infection by Mycobacterium tuberculosis.