Japan experienced its largest outbreak of highly pathogenic avian influenza (HPAI) during the 2022–2023 season, with 84 cases on poultry farms and the culling of more than 17?million birds. Commercial layer farms were the most affected, with 58 cases caused by the H5N1 subtype of the HPAI virus. To identify the risk factors and support future outbreak preparedness, a case–control study was conducted. All 58 affected commercial layer farms were designated as case farms, whereas 26 unaffected farms located within a 5?km radius of the case farms served as control farms. Data were collected through structured interviews using a questionnaire for the control farms and from on-site investigation records for the case farms. For control farms, all poultry barns on the premises were included, whereas only the initially affected barns were analyzed in the case farms. Logistic regression using a generalized linear mixed model was performed to evaluate the associations between HPAI outbreaks and explanatory variables related to farm- and barn-level factors, such as farm characteristics, husbandry and hygiene practices, barn structure, and environmental factors, including wildlife presence. Farm location was included as a random effect. The analysis identified a large number of laying hens on the farm (≥100,000 birds) as a potential risk factor. In contrast, changing shoes at the barn entrance and changing shoes or avoiding the use of the back door were identified as potential protective factors. These findings provide evidence-based guidance for strengthening biosecurity measures and improving HPAI prevention strategies on poultry farms in Japan.