Background: Highly pathogenic avian influenza (HPAI) remains a significant threat to poultry production in China. Layer specialized households (LSHs)—characterized by medium-scale operations (1000–15,000 birds)—are particularly vulnerable due to frequently suboptimal biosecurity measures. Cooperative prevention and control (CPC) strategies, including unified disinfection and joint monitoring, present a viable method for reducing HPAI risks. However, participation among LSHs remains low. Objectives: This study seeks to identify the key determinants influencing LSHs’ willingness to participate in CPC measures against HPAI, and further compare these driving factors across villages with versus without a documented history of HPAI outbreaks. Methods: A survey of 130 LSHs was conducted in two Chinese villages: Village A (with HPAI history) and Village B (without HPAI history). Data on socio-economic characteristics, production practices, and attitudes were collected via structured questionnaires. An Ordered Probit model was employed to analyze determinants of willingness to participate, measured on a 5-point ordinal scale. Results: Full-sample regression analysis demonstrated that older age, higher educational attainment, risk-tolerant attitudes, larger household scale, and higher annual household income exerted a significantly positive impact on the willingness to participate in the program. Surprisingly, greater knowledge of avian influenza exerted a significant negative effect. Regional subgroup analyses identified distinct drivers: in Village A (with HPAI history), prior experience of bird deaths positively influenced willingness, whereas longer breeding experience had a negative effect. In Village B (no HPAI history), frequent social interaction and greater breeding experience emerged as positive determinants. Conclusions: The findings underscore the critical role of regional context and specific household characteristics in shaping LSHs’ willingness to cooperate. Policy interventions to promote CPC should be regionally tailored: in previously affected areas, leveraging past outbreak experiences and ensuring equitable compensation is essential; in unaffected areas, fostering social networks and demonstrating CPC benefits through peer influence are more effective.