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Background  In higher plants, the  FAD2  gene encodes the microsomal oleate  Δ12 -desaturase, one of the key enzymes essential for the biosynthesis of the polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, in the biosynthesis of the polyunsaturated fatty acids (PUFAs) found in the cell membrane and cell wall, and it is thus of great importance to investigate the regulatory role of  FAD2  in anther development.    Results  We reported the molecular characterization of the cotton ( Gossypium hirsutum )  GhFAD2  gene family and the essential role of  GhFAD2–3  in cotton anther development.  G. hirsutum  contains four pairs of homoeologous  FAD2  genes ( GhFAD2–1  to  GhFAD2–4 ).  GhFAD2–3  is ubiquitously and relatively highly expressed in all analyzed tissues, particularly in anthers. Specific inhibition of  GhFAD2–3  using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the  GhFAD2–3  silenced plant ( fad2–3 ) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of the wild type (WT), the content of C18:1 was 41.48%, which increased by 5 fold in the  fad2–3  anther at the pollen maturation stage. Moreover, the ratio of monounsaturated to polyunsaturated fatty acid was 5.43 in  fad2–3  anther, which was much higher than that of the WT (only 0.39). Through compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating  GhFAD2–3  expression level to increase the oleic acid content.    Conclusions  Our work demonstrated the importance of C18:2 and/or C18:3 in the development of the pollen exine and anther cuticle in cotton and provided clue for further investigation of the physiological significance of the fatty acid composition for plant growth and development.    Electronic supplementary material  The online version of this article (10.1186/s12870-019-2010-9) contains supplementary material, which is available to authorized users.

GhFAD2–3 is required for anther development in Gossypium hirsutum