Background Equine dental diseases significantly impact on a horse’s overall health, performance, and quality of life. They can result in secondary infections and digestive disturbances, potentially leading to colic. A recently described disease affecting the incisors of horses is equine odontoclastic tooth resorption and hypercementosis (EOTRH). Understanding EOTRH is crucial for early diagnosis, effective management, and prevention of its severe consequences. Objectives To determine proteomic differences in incisor cementum in horses with and without clinical EOTRH. Study Design Comparative and observational clinical study. Methods Teeth were extracted and cementum was isolated using a diamond wire. Proteins were extracted using an optimised sequential workflow, and trypsin digested for mass spectrometry. Protein identification and label-free quantification was undertaken. Results In total 1149 unique proteins were detected in cementum across all samples. We identified four proteins exclusively in EOTRH affected cementum. EOTRH samples showed a higher heterogeneity than healthy samples. In total 54 proteins were increased in EOTRH, and 64 proteins were reduced (adjusted p-value < 0.05). Inflammatory proteins, such as cathepsin G (p = 0.004), neutrophil elastase (p = 0.003), bactericidal permeability-increasing protein (p = 0.002), azurocidin (p = 0.003) and lactotransferrin (p = 0.002) were all increased in EOTRH. Pathway analysis revealed that antimicrobial peptides (Z score 2.65, p = 1.93E-09) and neutrophil degranulation (Z-score 1.89, p = 1.7E-04) were commonly up-regulated canonical pathways. Main limitations The sample size was limited. Conclusion EOTRH leads to biochemical changes within the cementum proteome, which are important in explaining the physiological changes occurring in disease. Differentially abundant proteins may represent promising biomarkers for earlier disease detection and establishment of a cell-based model could provide further insight into the role these proteins play in hypercementosis.