Abstract: In this study, the different developmental stages of anthers cytological characteristics of Lycium barbarum male sterile line ′Ningqi No.5′ and fertile line ′Ningqi No.1′ were compared and analyzed by semi-thin section, and the transcriptomic profiles of anthers of ′Ningqi No.5′ and ′Ningqi No.1′ in microspore mother cell stage, tetrad stage and mononuclear stage were compared by RNA-seq technology. The results showed that the abnormal tapetal layer of the sterile line ′Ningqi No. 5′ began to appear in the tetrad period, and the callose wall outside the tetrad did not degrade, resulting in the inability to produce fertile pollen grains. A total of 2 619 differentially expressed genes（DEGs） were screened by transcriptome analysis at the three stages of anthers development, of which 1 609 were up-regulated and 1 010 were down-regulated, and 70 DEGs were found at all three different developmental stages. GO and KEGG analysis showed that these 2 619 DEGs are mainly involved in callose synthesis and degradation, lipid metabolism and flavonoid biosynthesis. The results of RT-qPCR were basically consistent with those of transcriptome sequencing. By analyzing the transcriptome DEGs, we identified 9 DEGs which may be related to callose synthesis and degradation, and 19 different-expressed transcription factors related to anther development. This study provides a new genetic resource and lay a foundation for further analysis of the molecular mechanism of male sterility in anther of Lycium barbarum.