Two human gastric cancer cell lines of differing invasive potential, SNU-484 and SNU-638 cells, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the eight cDNAs identified as being differentially expressed genes, it was determined that humbug, which encodes a truncated isoform of aspartyl (asparaginyl) beta-hydroxylase (AAH) missing catalytic domain, was overexpressed in highly invasive SNU-638 cells. Expression analysis showed that the mRNA expression level of humbug was correlated with invasive potential in various human gastric cancer cell lines. The forced expression of humbug to the human gastric cancer cell line AGS increased its anchorage-independent growth in 0.3% agar without affecting cell proliferation. Furthermore, humbug-transfected cells migrated more actively and showed an increased invasion rate relative to vector-transfectants or parental AGS in vitro. This is the first demonstration that humbug, a truncated form of AAH, can be overexpressed during the malignant progression of human gastric cancer cells and that it can function as a metastasis-inducing gene.