Discovering sugar metabolism genes is of great interest for lignocellulosic biorefinery. Xylose isomerases (XIs) were commonly screened from metagenomes derived from bovine rumen, soil, and other sources. However, so far, XIs and other sugar-utilizing enzymes have not been discovered from fecal metagenomes. In this study, environmental DNA from the fecal samples collected from yellow cattle (Bos taurus) was sequenced and analyzed. In the whole 14.26 Gbp clean data, 92 putative XIs were annotated. After sequence analysis, seven putative XIs were heterologously expressed in Escherichia coli and characterized in vitro. The XIs 58444 and 58960 purified from E. coli exhibited 22% higher enzyme activity when compared with that of the native E. coli XI. The XI 58444, similar to the XI from Lachnospira multipara, exhibited a relatively stable activity profile across different pH conditions. Four XIs were further investigated in budding yeast Saccharomyces cerevisiae after codon optimization. Overexpression of the codon-optimized 58444 enabled S. cerevisiae to utilize 6.4 g/L xylose after 96 h without any other genetic manipulations, which is 56% higher than the control yeast strain overexpressing an optimized XI gene xylA*3 selected by three rounds of mutation. Our results provide evidence that a bovine fecal metagenome is a novel and valuable source of XIs and other industrial enzymes for biotechnology applications.