Cellulose acetate (CA) is a semi-synthetic polymer obtained through the esterification of acetic acid with cellulose. CA is mainly used as industrial material to produce textiles, cigarette filters, and others. However, its use as an animal feed has not been evaluated. We focused on CA as a new cellulosic feed source for ruminant animals. In this study, a series of in vitro experiments were carried out to determine how CA addition affects rumen fermentation and microbiota. This sequencing data show ruminal microbiota changes by CA supplementation.