Abstractnanocrystalline cellulose (NCC) was prepared from cotton fiber by a single-step method under mild conditions using anhydrous phosphoric acid as the solvent. The absorbance peak of O-H was reduced, and the absorbance peaks of C=O and CH3 appeared in the Fourier transform infrared (FTIR) spectrum of the acetylated NCC with respect to that of the unmodified NCC. The roughly estimated degree of substitution was a little greater than 1.5 by FTIR analyses, implying that most of the free hydroxyl groups on the NCC surface were acetylated at 40 °C for 3 h. The carbons of the acetyl groups were clearly identified in the 13C cross polarization-magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) spectrum. The zeta potential was reduced from -32.12 mV to -20.57 mV after acetylation. Transmission electron microscope (TEM) and field-emission scanning electron microscope (FESEM) images showed that they were thread-like nano-crystals with a diameter less than 5 nm. Crystal structure analysis using X-ray diffraction (XRD) demonstrated that the acetylated NCC had the typical CelluloseⅡstructure. The PLA film reinforced with 3 wt% acetylated NCC content exhibited the highest tensile strength, which was increased by 117% compared to the control. SEM observation demonstrated good interfacial interaction between the acetylated NCC and the matrix.