Heart and neural crest derivatives expressed transcript 2 (HAND2) is a critical mediator of progesterone action in endometrial stromal cells. Silencing of Hand2expression in mouse uterus leads to an unopposed FGFR-mediated action that causes female mice infertility. To investigate the involvement of HAND2-FGFR signaling in pathogenesis of adenomyosis, immunohistochemistry, in situ hybridization, and quantitative real-time PCR were employed to assess gene expression in the normal endometrium, the paired eutopic endometrium and ectopic lesions obtained from women with adenomyosis. DNA methylation in the regions of HAND2promoter and the first exon was also monitored in these samples. Our results revealed that HAND2expression were dramatically reduced, but FGF9 expression and FGFR-ERK1/2-mediated MAPK signaling pathway were enhanced in the eutopic endometrium and ectopic lesions of patients with adenomyosis compared to the normal controls. Interestingly, expression of HAND2-AS1, a long noncoding RNA that resides adjacent to HAND2in genome, was also reduced in adenomyosis. DNA methylation analysis revealed that the bidirectional promoter between HAND2and HAND2-AS1, and the first exon of HAND2gene was heavily methylated in the eutopic endometrium and the ectopic lesions of adenomyosis. To investigate the regulation of gene expression by HAND2-AS1, HAND2-AS1expression was silenced in human endometrial stromal cells. In contrast to the downregulation of HAND2in response to HAND2-AS1silencing, FGF9expression was augmented significantly. Endometrial stromal cells lacking HAND2-AS1exhibited enhanced proliferation and migration potentials. Collectively, our studies revealed a new molecular mechanism by which HAND2-AS1is involved in the pathogenesis of adenomyosis via modulating HAND2-FGFR-mediated signaling.Loss ofHAND2-AS1 expression leads to downregulation of HAND2 expression and upregulation of FGFs secretion in endometrial stromal cells. EGFs then activate FGFR to ERK1/2-mediated MAPK signaling pathway, enhance endometrial cell proliferation and migration potentials in adenomyosis.Graphical Abstract