A xylanase derived from rumen ciliates designated as XynC, was successfully expressed and purified. The enzyme's catalytic properties, including its optimal pH (5.5) and temperature (40 °C), were determined, demonstrating high stability under mildly acidic and moderate thermal conditions. The substrate specificity of XynC was investigated by analyzing its hydrolysis pattern with beechwood xylan, where it preferentially degraded higher xylo-oligosaccharides (xylotetrose (X4), xylopentose (X5), xylohexose (X6)) while showing no activity on smaller oligosaccharides like xylobiose (X2) and xylotriose (X3). The in vivo effects of XynC on gut microbiota were evaluated by administering the enzyme to mice, followed by analysis of fecal 16S rRNA to assess shifts in gut bacterial populations. The results revealed dose-dependent modulation of gut microbiota composition, with a significant increase in Lactobacillus abundance observed in the medium- and high-dose groups, while no notable change was detected in the low-dose group. These dose-related microbial responses suggest that the efficacy of XynC in promoting probiotic growth is dependent on administration level. These findings suggest that XynC selectively promotes the growth of beneficial probiotics in a dosage-sensitive manner, supporting its potential as a functional feed additive for enhancing gut health and nutrient utilization in livestock.