Background <p>Periodontitis is a chronic inflammatory disease characterized by tissue destruction and immune dysregulation. While gingival crevicular fluid (GCF) serves as a non-invasive biomarker source, its molecular distinctions from periodontal tissue remain underexplored. This study conducted a comparative proteomic analysis of GCF and tissue samples from patients with Stage III–IV periodontitis, integrating differential expression, weighted gene co-expression network analysis, and protein–protein interaction networks to delineate compartment-specific molecular profiles and clarify their respective biological roles in periodontal pathophysiology.</p> Methods <p>Proteomic data were acquired from GCF and periodontal tissue using label-free LC–MS analysis. Differentially expressed proteins (DEPs) were identified using independent samples t-test (<i>p</i> &lt; 0.05, |fold-change| &gt; 2). WGCNA was performed to construct co-expression modules and identify functionally related protein clusters, followed by GO enrichment and protein–protein interaction (PPI) analyses using STRING and Cytoscape. Hub proteins were determined through CytoHubba according to centrality measures. Comparative analyses were conducted between tissue and GCF to define inflammation- and repair-related modules and to assess potential molecular interconnections between the two sample types.</p> Results <p>A total of 4,¹04 proteins were identified in periodontal tissue and 1,546 in GCF. Among these, 1,292 DEPs were detected in tissue and 280 in GCF. Periodontal tissue displayed coordinated upregulation of ribosomal proteins and collagen networks alongside mitochondrial components, indicating repair-oriented structural remodeling and metabolic activation. Conversely, GCF exhibited enrichment of neutrophil-derived immune effectors including MPO, ELANE, CTSG, S100A8, and apolipoproteins, representing innate immune activation. Network integration revealed that GCF and tissue maintained largely distinct molecular profiles with limited cross-compartment connectivity.</p> Conclusions <p>This comparative proteomic analysis demonstrates that periodontal tissue and GCF represent functionally distinct but complementary biological environments in periodontitis. Periodontal tissue exhibits enhanced structural and metabolic processes, whereas GCF predominantly reflects neutrophil-mediated immune responses. These molecular distinctions provide a basis for developing clinical, compartment-specific biomarker candidates that warrant analytical validation, thereby supporting precision-medicine-relevant diagnostic strategies in periodontal research.</p>