The importance of microRNAs at the post-transcriptional regulation level has recently been recognized in both animals and plants. We used the simple but effective sequential method of first Blasting known animal miRNAs against the horse genome and then using the located candidates to search for novel miRNAs by RNA folding method in the vicinity (+ -500 bp) of the candidates. Here, a total of 407 novel horse miRNA genes including 354 mature miRNAs were identified, of these, 75 miRNAs were grouped into 32 families based on seed sequence identity. MiRNA genes tend to be present as clusters in some chromosomes, and 146 miRNA genes accounted for 36% of the total were observed as part of polycistronic transcripts. Detailed analysis of sequence characteristics in novel horse and all previous known animal miRNAs were carried out. Our study will provide a reference point for further study on miRNAs identification in animals and improve the understanding of genome in horse.