Cellulose nanocrystal (CNC) is a sustainable biomaterial that has been used in many aspects of the food industry, but its effect on fat digestion and absorption is still underexplored. In this study, three CNCs were prepared from buckwheat bran. Their physicochemical properties were characterized, based on which the acetic acid-hydrolyzed CNC (ACCNC) with high absorption capacity was selected for the cytotoxicity evaluation and as a possible inhibitor for fat digestion and absorption in vitro and in vivo. ACCNC was proved to be nontoxic in the MTT assay and animal feeding tests. Especially, with the addition of ACCNC, the hydrolysis of fat was significantly reduced during the simulated digestion in vitro. In vivo testing also confirmed that ACCNC intake significantly reduced the elevated triglyceride, body weight, and fat accumulation levels. This study highlights the potential role of ACCNC prepared from buckwheat bran as an inhibitor for fat digestion and absorption.