We isolated a transposon-induced nonmotile mutant of the cyanobacterium Synechocystis sp. PCC 6803. The mutant was revealed to have a Tn5 insertion in the slr1443 gene that showed sequence similarity to a eukaryotic-type protein kinase. Thick pili were not observed on the mutant cell surface under the electron microscope. The slr1443 gene was not involved in transcription or translation of the pilA1 gene encoding pilin, the major component of thick pili. In the mutant, lower molecular mass pilin peptides were detected than in the wild-type. The pilin variant was not truncated at the N- or C-terminus of mature PilA1. The reduced molecular mass may have resulted from insufficient post-translational modification. The amounts of pilin variants were remarkably reduced in the periplasmic and surface fractions. The pilin variants were released into liquid media without being assembled into pili. Our finding suggests that Slr1443 plays an important role in pilus biogenesis at the level of the post-translational modification of pilin.