Abstract: In order to explore the molecular mechanism of sex differentiation process in Xanthoceras sorbifolium Bunge, flower buds at sex differentiation key stage were sampled to perform RNA-seq analysis. As a result, a total of 37.13 Gb raw data was obtained. After de novo assembly, a total of 29 430 Unigenes were produced with an average length of 1 144 bp, of which 21 887 Unigenes were functionally annotated. In addition, 4 864 differential genes were obtained. Compared with pre-sex differentiation stage（Ta）, 3 003 genes were up-regulated, and 1 861 genes were down-regulated. GO enrichment analysis showed that differential genes were mainly annotated to metabolic processes, plant hormone signal transduction, auxin-activated signaling pathways, and so on. KEGG enrichment analysis demonstrated differential genes were mainly annotated to biological functions such as plant hormone signal transduction, starch and sucrose metabolism. Six MADS-box related genes（TR576|c0＿g1, TR7438|c0＿g1, TR10037|c1＿g10, TR13325|c0＿g1, TR6316|c0＿g1, TR5807|c0＿g1） were screened involving stamen development, pollen tube growth, flower development regulation, petal development, process regulation of ovule development, transcription factor activity, and so on. The expression levels of 16 genes in two periods were detected by RT-qPCR, and the results were consistent with RNA-seq. The results of the present study would provide a reference for the research on the gene expression analysis in sex differentiation in X. sorbifolium.