Abstract: Skin, the largest organ in vertebrates, gives rise to skin appendages that evolve from specialized cells between the epidermis and dermis. Scales, a type of skin appendage, play crucial roles in fish by providing protection and reducing hydrodynamic drag. Current research on scale morphology and development primarily focuses on common cycloid scales. In contrast, the obscure puffer (Takifugu obscurus) exhibits extreme morphological diversity in its skin appendages, which manifest as prominent spines. However, the molecular mechanisms underlying spine formation in this species remain poorly understood. This study aims to preliminarily screen for genes involved in spine formation in T. obscurus through transcriptomic analysis, thereby providing a foundational dataset for further mechanistic investigation. We employed alizarin red staining to observe the distribution and morphological characteristics of the spines, followed by transcriptome sequencing of skin tissues from spiny and non-spiny regions to identify genes associated with spine formation. Staining revealed that the spines are highly mineralized structures, each consisting of a basal plate embedded in the dermis and an exposed sharp spine, distributed primarily across the dorsal, ventral, and lateral body surfaces. Morphological observations showed slight regional variations: dorsal spines tended to be shorter and thicker, while ventral spines were longer and thinner. Transcriptome sequencing identified a total of 1,815 significantly differentially expressed genes (DEGs), of which 1,456 were upregulated and 359 were downregulated. Enrichment analysis further revealed that these DEGs were significantly enriched in pathways such as the PI3K-Akt signaling pathway, Focal adhesion, and ECM-receptor interaction. Further screening identified multiple genes closely associated with spine formation, including members of the collagen gene family, members of the secreted calcium-binding phosphoprotein (SCPP) gene family (e.g., scpp1), members of the integrin gene family (e.g., itga4, itga8, itga10), as well as tgfb3 and tmem9.These findings provide data support for elucidating the mechanism of spine formation in T. obscurus by identifying relevant genes and signaling pathways, thereby laying a foundation for further studies. It fills a data gap in the study of specialized skin appendages in tetraodontiform fishes and contributes to understanding the developmental mechanisms and evolutionary processes of different skin appendages in vertebrates.