Aims Campylobacter jejuni is a major cause of foodborne gastroenteritis. We previously reported the widespread Camp.jejuni sequence type (ST)-4526 in Japan from 2005 to 2006. This study assesses the potential for this genotype to thrive thereafter. Methods and Results Fifty human Camp.jejuni isolates collected in 20102011 in Osaka, Japan, were genotyped by multilocus sequence typing (MLST). This approach identified 22 STs and 11 clonal complexes (CCs), including four novel STs. A comparative analysis to the previous data set showed the predominance of CC-21, in which ST-4526 and ST-4253 represented 39 and 63% in each of the two time frames, indicating their continued widespread presence. These two STs belong to close evolutionary lineages and are also isolated from chicken meat. The superior abilities of ST-4526/ST-4253 representatives to colonize chicken gut were demonstrated by co-infections with ST-21, ST-50 and ST-8 representatives. Conclusions Data provide evidence for the continued widespread of ST-4526/ST-4253 among human clinical isolates in Japan. These STs showed adaptive fitness to chicken. Significance and Impact of the study This is the first evidence of the continued thriving of ST-4526/ST-4253 in Japan with their increased in vivo fitness. Our findings suggest that poultry mediates the microevolution of this pathogen, thereby enabling these STs to become widespread.