Hyaluronan (HA) has been extensively used for various medical applications, including osteoarthritis, tissue augmentation and ocular surgery. More recently, it has been investigated for use in polymer therapeutics as a carrier for drugs and biologically active proteins, thanks to its biodegradability, biocompatibility and inherent biological properties. Such biological functions are strongly dependent on HA's chain length, yet the molecular weight of HAs used in polymer conjugates varies widely and is inconsistent with its intended application. Therefore, this study aimed to determine the ideal chain length of HA to be used in polymer conjugates for enhanced tissue repair. HA fragments (M(w) 45,000-900,000g/mol) were prepared by acid hydrolysis of rooster comb HA and their physicochemical and biological properties were characterized. Such HA fragments had a highly extended, almost rod-like solution conformation and demonstrated chain length- and concentration-dependent viscosity, while exposure to HAase caused a rapid reduction in HA viscosity, which was most significant for the native HA. Initial HA hydrolysis rate by HAase varied strongly with HA chain length and was dependent on the formation of a stable enzyme-substrate complex. When normal human dermal fibroblasts were exposed to the different HA fragments for 72h, only native (900,000g/mol) HA reduced proliferation at 1000μg/mL. Conversely, only the smallest HA fragment (70,000g/mol) reduced the proliferation of chronic wound fibroblasts, at 1000μg/mL. The 70,000g/mol HA fragment also promoted the greatest cell attachment. These observations demonstrate that low molecular weight (70,000-120,000g/mol) HA fragments would be best suited for the delivery of proteins and peptides with applications in chronic wound healing and paves the way for the rationalized development of novel HA conjugates.