The intracellular pH is regulated by a delicate balance of ion distribution across the plasma membrane and the physico-chemical properties of intra- and extracellular components. We analyzed the effects of glycosaminoglycans on the intracellular pH of fibroblasts by using the fluorescent pH indicator BCECF-AM. Addition of hyaluronan, hyaluronan oligosaccharides, chondroitin sulfate, or heparin to the culture medium of fibroblasts caused intracellular acidification from pH 7.2 to 6.7 in a concentration dependent manner. High molecular weight hyaluronan acidified more than hyaluronan oligosaccharides at the same concentrations. Hyaluronidase treatment or inhibition of hyaluronan export with xanthohumol led to intracellular alkalization. These observations indicated that extracellular glycosaminoglycans participate in intracellular pH regulation. The mechanism was explained by Donnan effects and molecular crowding. J. Cell. Physiol. 228: 2071–2075, 2013. © 2013 Wiley Periodicals, Inc.