Physically cross-linked hydrogels from hyaluronan (hyaluronic acid, HA) were prepared by a freezethaw technique at low pH. The effect of the freezingthawing of HA solutions on the formation of physical cryogels is typical for the processes of noncovalent cryostructuration that takes the advantages of mild fabrication conditions and the absence of organic solvents and toxically cross-linking agents. The effects of processing steps (freezing time and number of freezethaw cycles), HA molecular weight (M-w), and the addition of typical polycarboxylic and polyhydric small molecules such as dicarboxylic acids and polyols on the formation of HA cryotropic hydrogels were investigated. Results verified that long freezing time and repeated freezethaw cycles benefited the alignment of polymer chains in the unfrozen liquid microphase, thereby promoting the formation of intermolecular aggregations and dense fibrillar network structures. High M-w of HA endowed the cryogel with strong mechanical strength. The influences of various small molecules on the cryogelation of HA revealed the different intermolecular association patterns in the gel network. Both succinic and glutaric acids participated in HA cryogelation, whereas oxalic, malic, and tartaric acids as well as some polyols (glycol, butanediol, and glycerol) inhibited the cryostructuration of HA. Hydrogen bonding and intermolecular interactions in acidic cryogels and in neutral cryogels obtained by in situ neutralizing the acidic cryogel were discussed at the molecular level in correlation with intermolecular associations and molecular conformation. A gelation mechanism for HA cryogel was proposed. In addition, experimental findings showed that the neutral HA cryogels possessed enhanced thermostability, resistance to acid decomposition, and enzyme degradation which are essentially important properties for biomaterials.