Synopsis Background Fibroblasts are an important component of the skin determining its properties. N-Acetylglucosamine (NAG) is the substrate for hyaluronan synthesis, and it also has anti-inflammatory and anti-senescent activity in mesothelial cells. Methods We tested in in vitro-cultured human skin fibroblasts how supplementation of culture medium with NAG 10mmolL(-1) changes properties of these cells. Results Fibroblasts cultured in presence of NAG produced more proteins and that was mainly due to increased synthesis of collagen (+33% vs. control, P<0.05). Hyaluronan synthesis was increased (+107% vs. control, P<0.001), but interleukin-6 synthesis was reduced (-22% vs. control, P<0.05). Fibroblasts cultured in medium with NAG 10mmolL(-1) demonstrated improved ability to heal the injured layer of cells (+34% vs. control, P<0.05). Additionally senescence of fibroblasts undergoing replicative ageing in the presence of NAG was less pronounced, as reflected by smaller increase in the population doubling time (-70% vs. control, P<0.05). Conclusion We conclude that NAG induced changes in the skin fibroblasts' properties maybe important for prevention of the age-dependent changes in its structure and function.