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Cluster of differentiation 44 (CD44), a marker for cancer stem cells, has been reported to be associated with poor prognosis in non-small cell lung cancer (NSCLC), but its involvement in tumor growth has not fully been elucidated. The present study explored the associations between CD44 expression and clinicopathological features using immunohistochemistry in 94 NSCLC cases, as well as proliferation in cells with aberrant expression of this protein. Overexpression of CD44 was achieved by transfecting H1299 cells with CD44 expression vectors, and inhibition of CD44 expression was performed by transfecting small interfering RNAs into A549 cells. Cell proliferation was measured using Cell Counting Kit-8 assays and flat plate colony formation assay was performed to confirm the cellular anchorage growth <i>in vitro</i>. In total, 64 (68.1%) of the 94 NSCLC cases stained positive for CD44 expression. High expression of CD44 was associated with advanced T stage in NSCLC. Overexpression of CD44 in H1299 cells promoted cell proliferation and colony formation <i>in vitro</i>. Meanwhile, knockdown of CD44 expression in A549 cells suppressed cell proliferation and colony formation <i>in vitro</i>. High expression of CD44 may promote NSCLC progression by increasing cancer cell proliferation; therefore, it may serve as a potential biomarker for diagnosis or target therapy.