Pilus-mediated motility is essential for the optimization of photosynthesis and environmental adaptation in the cyanobacterium Synechocystis sp. PCC 6803 (Syn6803). To identify the genes required for pilus-mediated motility in Syn6803, we applied a forward genetic approach using a Tn5 mutant library and reverse genetics using interposon mutagenesis. One of the identified genes, sll0899, bears sequence similarity to acyltransferases and nucleotidyltransferases. The sll0899 gene product is not involved in the transcription or translation of pilA1, which encodes pilin, the major component of pili. Instead, the sll0899::Cm(r) mutant produced pilins with increased molecular mass, suggesting the existence of different PTMs. Using MS, we found that the wild-type (WT) and mutant pilins were glycosylated between amino acids 67 and 75. Analyses by quantitative MS and high-pH anion exchange chromatography (HPAEC) revealed that the glycan in WT pilin is composed of xylose and fucose, whereas an additional sugar, rhamnose, was found in the glycan of sll0899::Cm(r). Our findings suggest that an alteration in the O-linked glycan of pilin is responsible for the loss of pilus-mediated motility in sll0899::Cm(r).