As endogenous small RNAs in plant, miRNAs can play important roles in pollen development and fertility regulation. In order to provide theoretical basis for further discussing miRNAmediated regulatory mechanism of male sterility in broccoli, we constructed the sRNA libraries of cytoplasmic male sterile line and its maintainer line by highthroughput sequencing in this study. Meanwhile, the expression characteristics of fertility related miRNAs and their predicted target genes were detected by qRTPCR technology. The results showed that: (1) a total of 181 conserved and 8 novel miRNAs were identified in broccoli. Among them, 47 miRNAs were differentially expressed. 24 known miRNAs and 4 novel miRNAs were upregulated and the rest were downregulated. Two differentially expressed miRNAs were only expressed in sterile line, and seven differentially expressed miRNAs were specific to its maintainer line. (2) Bioinformatics analysis showed that two associated miRNAmRNAs ［miR8591 targeted pollen coat gene (T2/Unigene_BMK.21608) and miR5174e1 regulated amino acid permease gene (T2_Unigene_BMK.31772)］ were identified. These two miRNAs could negatively regulate their corresponding target genes and participate in microspore development and male sterility occurrence in broccoli. (3) qRTPCR analysis showed that the expression levels of miR159a1, miR164a1, miR319a1 and miRn11 in male sterile line were higher than those in maintainer line. The expression levels of miR164a1, miR319a1 and miRn11 decreased with development of flower bud in sterile line, but increased in maintainer line; miR319a1 and miR397a2 were high expressed in stamen and low in other parts of flower buds, and the expression level of miR397a2 in sterile line was significantly lower than that in maintainers line. Our study speculated that miRNAs may lead to the occurrence of male sterility by inhibiting the expression of male sterility related PPR gene, regulating the expression of LAC gene and regulating hormone and Ca²⁺ mediated signal transduction pathway in broccoli.