We treated the rat thyroid follicular epithelial cell line FRTL-5 with LPS to establish an in vitro cell model mimicking Hashimoto’s thyroiditis. Subsequently, the conditioned medium derived from LPS-stimulated FRTL-5 cells was collected and used to co-culture rat pancreatic β-cell line INS-1. Finally, the INS-1 cells after co-cultivation were harvested for proteomic sequencing analysis.