The razor clam Sinonovacula constricta inhabits sulfide-rich intertidal sediments and exhibits remarkable tolerance to this toxicant, yet the role of its periostracum layer in sulfide adaptation remains poorly understood. In this study, we investigated the composition, structure, and sulfur-responsive expression of periostracum proteins. Scanning electron microscopy and energy-dispersive X-ray spectroscopy revealed that the periostracum layer is approximately 10 μm thick and contains 1.43 wt% sulfur, suggesting its involvement in sulfur deposition. Using LC-MS/MS, we identified 77 high-confidence proteins from the periostracum, which were classified into six functional categories: enzymes, framework proteins, immune-related proteins, calcium ion-related proteins, other proteins, and proteins with unknown functions. Phylogenetic analyses of representative proteins revealed bivalve-specific evolutionary patterns, with several proteins exclusively present in Bivalvia. Quantitative PCR analysis under sulfide exposure (10 μM and 100 μM Na₂S) showed that genes encoding chitin-binding protein 3 and tyrosinase were significantly upregulated in the mantle, whereas collagen and cadherin 3 were generally suppressed. These results indicate that the periostracum layer is not merely a passive physical barrier but a dynamic biological interface that responds to sulfide stress through coordinated regulation of structural and enzymatic proteins. This study provides the first comprehensive proteomic profile of the razor clam periostracum and highlights its potential role in sulfide tolerance, offering insights for sulfur-tolerant aquaculture breeding and bioremediation applications.