The fission yeast Schizosaccharomyces pombe exhibits invasive growth and nonsexual flocculation in response to nitrogen limitation. Gsf2, a flocculin of fission yeast, is required not only for nonsexual flocculation but also for invasive growth through the recognition of galactose residues on cell surface glycoconjugates. We found that pyruvylation negatively regulates nonsexual flocculation by capping the galactose residues of N-linked galactomannan. We investigated whether pyruvylation also regulates invasive growth. The pvg4(+) gene originally was isolated as a multicopy suppressor of a pvg4 mutant defective in the pyruvylation of N-linked oligosaccharides. However, we did not detect a defect in cell surface pyruvylation in the pvg4/mbx2 deletion mutant, as assessed by alcian blue staining and a Q-Sepharose binding assay. Instead, the deletion prevented invasive growth under conditions of low nitrogen and high glucose, and it reduced the adhesion and flocculation of otherwise flocculent mutants by reducing gsf2(+) expression. mbx2(+)-overexpressing strains exhibited nonsexual and calcium-dependent aggregation, which was inhibited in the presence of galactose but mediated by the induction of gsf2(+). These findings indicate that Mbx2 mediates invasive growth and flocculation via the transcriptional activation of gsf2(+) in fission yeast. In addition, we found that fission yeast Mbx2 induces the nonsexual flocculation of budding yeast by the activation of FLO1.