Na(+)/H(+) exchanger 1 (NHE1), a primary regulator of intracellular pH (pHi) and extracellular pH (pHe), plays a significant role in acidifying the tumor microenvironment, possibly resulting in their malignant potential. However, currently, very little is known about the roles of NHE1 in invasion of hepatocellular carcinoma (HCC) cells. We have recently shown that NHE1 is over-expressed in HCC tissues and that this increased expression is associated with HCC invasiveness. In this study, we also found that NHE1 is over-expressed in HCC cell lines. Subsequently, we silenced NHE1 expression in the human HCC cell line SMMC-7721 using RNA interference (RNAi) and examined the invasiveness and proliferation of NHE1-silenced SMMC-7721 cells and the matrix metalloproteinase-2 (MMP-2) activity. The knockdown of NHE1 expression significantly inhibited the invasive ability of SMMC-7721 cells but had only a minor effect on the cellular proliferation rate. Moreover, NHE1 knockdown significantly reduced the secretion of MMP-2. Further experiment using amiloride (an inhibitor of NHE1) confirmed the above result. Together, these findings indicate that NHE1 has an important role in SMMC-7721 cell invasion and that NHE1 might be a new target of HCC treatment.