© 2022, Veteriner Fakultesi Dergisi. All rights reserved.The study aimed to determine the effect of xylitol, added to the dry period total mix ration (TMR) of dairy cattle, on in vitro rumen fermentation, total gas production, methane production, estimated digestion values, organic acids and ammonia-nitrogen contents and the number of total protozoa. Xylitol was added to dairy cattle TMR at different rates (0%, 2% 4%, and 8%; dry matter basis). The xylitol supplementations at 2 and 4% did not affect in vitro total gas production, in vitro methane production, metabolizable energy (ME) and organic matter digestibility (OMd) values (P>0.05). However, 8% xylitol supplementation decreased in vitro total gas production, in vitro methane production, ME and OMd (P<0.05). The molarities of total volatile fatty acid (TVFA) percentages of acetic acid (AA), propionic acid (PA) and butyric acid (BA) in TVFA, ammonia-nitrogen concentration, and the number of total ciliate protozoa of the in vitro rumen fluid of xylitol supplementations at 2%, 4%, and 8% were similar to those of control TMR (P>0.05). The iso-valeric acid (IVA), iso-butyric acid (IBA) and valeric acid (VA) percentages in TVFA of in vitro rumen fluid linearly decreased with xylitol supplementation, especially 8% xylitol supplementation (P<0.05). Besides, 2% and 4% xylitol supplementations to dairy cattle TMR numerically increased the concentration of ammonia-nitrogen and the number of total ciliate protozoa in in vitro fermentation fluid (P>0.05). Consequently, the supplementation of 2% and 4% xylitol to dairy cattle ration did not affect the in vitro rumen fermentation (total gas production, methane production, estimated digestion values, organic acids and ammonia-nitrogen parameters). However, 8% supplementation xylitol to dairy cattle ration had the potential to affect the before-mentioned in vitro ruminal fermentation parameters adversely.