<p>Honey bees are economically important pollinators and widely studied model organisms. The phenomenon of honey bee caste differentiation manifested by ovary development has been a hot topic in apicultural research. Juvenile hormone (JH) has a critical role in caste differentiation in honey bees. Here, we analyzed the morphology and transcriptome changes in honey bee ovary after knocking down the expression of the JH answer gene <i>AmKr-h1</i> by RNAi. As a result, the number of ovarian tubes was significantly reduced in the RNAi group and a total of 920 differentially expressed genes (DEGs) were found between the ovaries of the RNAi and negative control (NC) groups. Many of these genes are closely related to honey bee caste differentiation. In addition, 109 differentially expressed alternative splicing events (DEASEs) associated with 97 genes were identified between the RNAi group and NC group. The results of this study demonstrate that <i>AmKr-h1</i> significantly influences ovarian caste differentiation and gene expression in honey bees. Our results offer gene expression data that deepen the understanding of the molecular mechanisms underlying ovarian differentiation between queens and workers.</p>