NK4 suppresses invasion and metastasis of tumor cells by means of dual actions as HGF antagonist and angiogenesis inhibitor. Our previous studies showed that NK4 suppresses the implantation of tumor cells to the peritoneal milky spots (MS) by intraperitoneal injection (i.p.) of adenovirus vector expressing NK4 (Ad-NK4) or NK4 gene-transfected tumor cells. In the present study, we investigated the antitumor mechanisms of NK4 in the suppression of peritoneal implantation. When evaluated by a fluorescent microscopy, a prior injection of Ad-NK4 suppressed peritoneal implantation immediately after the injection of GFP-expressing tumor cells. DNA microarray analyses also demonstrated a reduced expression of some adhesion molecules in NK4 gene-transfected tumor cells as compared to neomycin gene-trasfected cells (control). In the in vitro adhesion assay, the adhesion to some types of the extra cellular matrixs (ECM) was significantly decreased in NK4 gene-transfected cells as compared to the control. These results suggest that NK4 may suppress peritoneal implantation by inhibiting adhesion of tumor cells to ECM around MS.