Recent studies show that the vacuolar-type H+-ATPase (V-ATPase) 16 kDa subunit is expressed on plasma membrane of cancer cells. We hypothesized that V-ATPase 16 kDa subunit is directly involved in cell invasion. In the present study we established transfectants overexpressing V-ATPase 16 kDa subunit at the mRNA level, and found that these transfectants showed an enhanced invasiveness through matrigel with a concomitant increases in secretion of matrix metalloproteinase-2. Moreover, antisense oligonucleotides of the V-ATPase 16 kDa subunit suppressed invasive human A549 cell invasion with concomitant decreases in secretion of matrix metalloproteinase-2. The results suggest that the V-ATPase 16 kDa subunit is directly involved in cell invasion and that matrix metalloproteinase-g is responsible for promoting the invasion by the V-ATPase 16 kDa subunit.