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In recent years, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to play important roles in tumor biological function. The aim of this study was to investigate the diagnostic and prognostic value of lncRNA H19 and miR-21 expression in non-small-cell lung cancer (NSCLC). H19 and miR-21 expression was measured in tumor tissues and corresponding non-tumor lung tissues from 200 patients by quantitative reverse transcription polymerase chain reaction. Moreover, the in vitro and in vivo effects of H19 knock out in A549 cells were investigated. Expression of both H19 and miR-21 was significantly higher in lung tissues from patients with NSCLC than in normal lung tissues. Increased expression of H19 and miR-21 was positively correlated with advanced tumor-node-metastasis stage and tumor size. miR-21 expression was highest in stage I and II NSCLC, whereas H19 expression was highest in stage III and IV NSCLC. Knockout of H19 significantly inhibited NSCLC cell proliferation both in vitro and in vivo. The results show that H19 may mainly contributes to the progression of NSCLC, and its expression levels can reflect the invasive and metastatic status to some extent. miR-21 expression more likely plays an important role in early stage NSCLC. Moreover, H19 and miR-21 interact in the regulation of NSCLC, and with greater expression of both H19 and miR-21, overall survival decreased. The combination of H19 and miR-21 may have diagnostic value in NSCLC and represent a target for new NSCLC treatments.