Polyurethane/crystalline nanocellulose (PU/CNC) nanocomposites were fabricated through in-situ polymerization by using polydimethylsiloxane (PDMS), methylene diphenyl diisocyanate (MDI), and 1,4-butanediol (BDO) as polyol, aromatic diisocyanate, and chain extender, respectively. CNC content affected the microstructure and thermophysical properties of the nanocomposites. Incorporation of CNCs into PU affected the interactions between soft and hard segments. Although CNCs had slight effect on thermal degradation of nanocomposites, they significantly affected thermophysical properties. For example, CNCs decreased the crystallization of hard segment and glass transition temperatures of hard and soft segments were shifted to higher temperatures. PU showed a relatively high toxicity against human fibroblast cells because of presence of aromatic segments in its structure. Although cytocompatibility was improved by addition of CNCs, PU/CNC nanocomposites showed relatively cytotoxic behavior at higher concentrations.