Autoagglutination (Agg) ofBordetella pertussisis often observed in clinical laboratory. However, its causal factors and frequency in circulating strains are unknown. Repeated single colony isolation enabled us to detect an Agg-mutant in the supernatant of an Agg+strain ofB. pertussis. Whole-genome sequencing and immunoblot analysis disclosed that the Agg-mutant had a single C-deletion in itsfim3promoter region (Pfim3) which abolished Fim3 fimbriae production. AB. pertussis fim3-knock out mutant also lacked the Agg+phenotype. Agg+clinical isolates were detected a higher production of Fim3 than Fim3-producing Agg-isolates.B. pertussisis known to harbor multiple Pfim3 poly(C) lengths within a single strain culture and our newly developed PCR/LDR assay revealed that Agg+isolates harbor the highest Pfim3 poly-14C abundance. We evaluated the frequency of autoagglutination in clinicalB. pertussisisolates collected in Japan between 1994 and 2018 (n = 203). Fim3 production was confirmed for 190 isolates and 74.7% of them displayed the Agg+phenotype. The Agg+phenotype was strongly associated with Pfim3 poly-14C abundance. Taken together, our findings demonstrated thatB. pertussisautoagglutination occurs in response to high Fim3 levels and the Agg+strain has predominated in Japan over the past two decades.