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A flexible chitin nanofiber (ChNF) film with a thin fiber morphology, named, scaled-down (SD)-ChNF film, was previously found to be formed via successive partial deacetylation of the parent self-assembled ChNFs, cationization/dispersion via electrostatic repulsion in aqueous acetic acid, and suction filtration/drying. In this study, acetylation of a SD-ChNF film using acetic anhydride in pyridine was carried out to improve the mechanical properties. The FT-IR spectra of the acetylated SD-ChNF films suggested that acetylation progressed from the surface to the interior of the films with the increasing amounts of pyridine and elevating temperatures. The degrees of acetylation (DA) strongly affected the chitin crystallinity and surface morphology of the acetylated SD-ChNF films. Tensile testing of the acetylated SD-ChNF films indicated that the mechanical properties were improved by adjusting the DA values of the films. For example, the acetylated SD-ChNF film with an 1.84 DA value on surface showed values of 44.1 MPa and 24.9% for tensile strength and elongation at break, respectively. [ABSTRACT FROM AUTHOR]