Introduction Sleep fragmentation (SF) is a hallmark of sleep disorders and has been associated with systemic health issues, but its specific impact on skin health remains unclear. This study aimed to investigate whether SF impairs skin barrier function and identify the biological pathways involved in SF-induced skin damage. Methods Twenty-four 6-week-old male BALB/c mice were divided into home cage control (HC) and SF groups. SF was induced using a commercially available SF chamber. Skin barrier function was assessed by measuring transepidermal water loss (TEWL) at 4 and 8 weeks. Epidermal thickness and dermal collagen density were also measured. Total RNA sequencing (RNA-Seq) and bioinformatics analysis were conducted to identify the affected pathways. Results TEWL was significantly higher in the SF group than in the HC group at 8 weeks. Epidermal thickness and dermal collagen density were significantly lower in the SF group than in the HC group. In the SF group, 133 differentially expressed genes were identified, of which 14 were upregulated and 119 were downregulated. RNA-Seq and bioinformatics analysis revealed an altered fatty acid metabolism pathway in the skin of mice subjected to chronic SF. This was validated through quantitative real-time polymerase chain reaction. Conclusion SF caused physiological and histological changes in the skin, altering the fatty acid metabolism pathway. The role of this pathway in SF-induced skin damage requires further exploration.