The anther is one of the most vulnerable organs to temperature stress. Many previous works focused on the genes regulating anthers development, but few results of miRNA in anther development were reported. In order to investigate the transcriptional regulation of temperature-sensitive anther development, RNA-Sequencing was used to study micRNA in anthers of Arabidopsis thaliana under 16 degrees C and 27 degrees C. A total of 46.26 million clean reads were generated and mapped to 715,748 small RNA sequences containing 281 miRNAs. Then 13 differentially expressed (DE) miRNAs, containing 3 novel miRNAs were found. Comprehensive analysis of miRNA expression showed 7 miRNAs were down-regulated and 6 miRNAs were up-regulated. Furthermore, 13 DE miRNAs putatively regulated 614 DE mRNAs. Among them, 20 important anther genes were predicted as target genes of MIR319A, MIR447A, MIR4478 and MIR3988, respectively. Over-expression MIR319A and MIR447A could effectively inhibit the transcription of target genes and lead to male sterile. It suggested that DE miRNAs might mediate temperature signals and regulate anther and pollen development. Our work will provide a broader idea and valuable data information for further understanding the mechanism of thermo-sensitive male fertility in plants. (C) 2020 Elsevier Inc. All rights reserved.