Abstract:[Background] As the resistance of pathogens to antibiotics is enhancing, it is increasingly important to develop safe and reliable antibiotic alternatives in the animal husbandry. Probiotics with no toxicity, no residues, and low costs have emerged as potential solutions, offering effective supplements of beneficial microorganisms in the intestinal tract. [Objective] To isolate probiotics from the intestine of sheep. [Methods] The intestinal contents and feces of healthy sheep were cultured and purified with the MRS medium, and the isolates were then identified by morphological analysis and 16S rRNA gene sequencing. The probiotic properties of the isolates were assessed, including the antimicrobial activity, acid and bile salt tolerance, self-aggregation capacity, antibiotic sensitivity, and safety in mice. [Results] Several strains were isolated from the intestine of healthy sheep, among which A12 and C1 were identified as Enterococcus mundtii and E. faecium, respectively. The cell-free supernatants of isolates A12 and C1 had strong inhibitory activities against Staphylococcus aureus and Salmonella choleraesuis. The isolates A12 and C1 had good acid and bile salt tolerance and strong self-aggregation ability and were susceptible to a variety of antibiotics. In addition, the two isolates did not induce obvious pathological lesions in mice, demonstrating good safety. [Conclusion] E. mundtii strain A12 and E. faecium strain C1 isolated from the sheep intestine have excellent probiotic properties and good safety, showcasing the potential to be developed as probiotics.