Abstract: Objective: To investigate the role of CD44v3 in adhesion and invasion behavior of human lung carcinoma cell A549 induced by hyaluronan in vitro. Methods: The RNA interference plasmid expressing shRNA (short hairpin RNA) against CD44v3 was designed and constructed, and was used for transfection of the A549 cell line. The expression of CD44v3 before and after transfection of the A549 cell line was examined by the reverse transcription polymerase chain reaction (RT-PCR) and western blot methods. Both Adhesive and invasive ability before and after transfection of the A549 cell line were determined by the plate adhesion model and Boyden chamber model. Results: Compared to pre-transfection, the A549 cell CD44v3 mRNA and expression of proteins transfected with RNA interference plasmid as well as the cell population sticking to the flat plate and penetrating septum of Boyden chamber decreased significantly (P<0.01). Conclusions: CD44v3 mediates the in vitro adhesion and invasive behavior of hyaluronic acid-induced human lung carcinoma cell line A549.