In higher plants, microRNA (miRNA) is involved in regulation of developmental processes, including sexual organ development. Seven novel miRNA families with one known miRNA were isolated by constructing a small RNA library from a mixture of anther from a cytoplasmic male sterile line and its maintainer. Two miRNAs are conserved in plant species. A total of 18 potential targets were identified for the eight miRNA families, including 15 proteins annotated with function and three unknown proteins. The known proteins include several proteins relevant to cell structure and stress response, transcription factors, and enzymes associated with metabolic and signaling pathways, playing important roles in microspore development. Quantitative real-time PCR assay revealed different expression patterns of the miRNAs between the cytoplasmic male sterile line and its maintainer. Each of the miRNAs tended to be down-regulated after the tetrad stage in a fertile line. However, most of the miRNAs in the cytoplasmic male sterile line were shown to be up-regulated from the tetrad to mononuclear stage, displaying special expression patterns differing from the ones in fertile line. We conclude that additional inactive miRNA pathways are essential during pollen development for a fertile line to ensure male fertility. Contrarily, miRNAs are up-regulated during the period from the tetrad to mononuclear stage, which contributes to pollen abortion for a cytoplasmic male sterile line.