The structural changes of the glucopyranose chain and the chemical compositional response of cellulose nanofibers (CNFs) under thermal exposure (at 190 °C for 5 h) have remained a significant gap in the understanding of the long-term performance of nanocellulose. Herein, CNF films with different chemical compositions were investigated to confirm the structural transformation of glucopyranose (coupling constant of OH groups changed up to 50%) by nuclear magnetic resonance (NMR) analysis. Remarkably, the glucopyranose rings underwent partial dehydration during the thermal exposure resulting in enol formation. This study confirms the chain mobility that could lead to the conformational and dimensional changes of the CNFs during thermal exposure. The broad range of conformations was defined by the dihedral angles that varied from ±27° to ±139° after thermal exposure. Investigation into the mechanism involving chemical transformation of the substrates during heating is important for the fabrication of the next generation of flexible electrical materials.