Background: Glucosamine sulphate (GS) is essential in the biosynthesis of glycolipids, glycoproteins, glycosaminoglycans (GAGs), hyaluronate, and proteoglycans. Connective tissues primarily contain collagen and proteoglycans and play an important role in skin ageing. Objective: The objectives were to assess ex vivo the impact of GS on skin ageing parameters and in vivo the effect of GS on the skin physiology of mature healthy volunteers after oral intake. Methods: The impact of GS on skin ageing was assessed ex vivo via different immunohistochemical assays and histology and via a clinical study using biopsies. Modulation of selected skin physiology markers was assessed by real-time quantitative PCR on skin punch biopsies obtained from 8 healthy >50-year-old women having ingested GS 250 mg once daily for 8 weeks. Results: Ex vivo, GS significantly (all p ≤ 0.02) increased the expression of CD44 and collagen type IV, the epidermis GAG level, and collagen type I synthesis. After 8 weeks of oral GS administration, a significantly increased expression was observed at the mRNA level for vimentin, fibromodulin, biglycan, xylosyl transferase, hyaluronan synthase, collagen types I and III, bone morphogenic protein-1, and decorin (all p ≤ 0.05). Conclusion: Both experiments showed that GS has a positive effect on epidermal and dermal markers associated with age.