Cell surface structure plays a key role in Microcystis colony formation. The S-layer is a crystalline array of monomolecular proteins that constitute the outermost component of the cyanobacterial cell envelope. To date, no biochemical characterization of the S-layer protein in Microcystis has been reported. Here, we compared S-layer on the cell wall of the unicellular strain Microcystis aeruginosa PCC7806 with the colonial strain M. aeruginosa XW01. We observed crystalline S-layers in XW01 cell walls; however, similar structures were not observed in PCC7806. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed a thick putative S-layer protein band with an apparent molecular weight of 70 kDa extracted from XW01 cells, as well as an S-layer peptide fragment with the sequence ETYPLLAAPGAATDATR, similar to the translated product from PCC7806 unknown gene CAO89090.1. The amino acid composition of the translated CAO89090.1 product shared biochemical characteristics with those of bacterial S-layer proteins. Furthermore, a 1002-bp DNA fragment amplified from XW01 displayed 95% similarity with the CAO89090.1 gene, while the S-layer gene expression in XW01 was 36-fold higher than that observed in PCC7806. These data suggested that the S-layer protein plays a key role in Microcystis colony formation due to its significant contribution to cell surface hydrophobicity.