Research on abortion and the mechanism of Cucurbita pepo L male infertility is of great value for promoting breeding and hybrids production. Taking the male sterile plants (X-MS) and fertile plants (X-MF) of zucchini as test materials, through transcriptome sequencing, as well as the determination of endogenous hormone contents, antioxidant enzyme activities, sucrose and starch contents, the results showed that from the tetrad stage (T1), early uninucleate stage (T2), middle to late uninucleate stage (T3), binucleate stage (T4) to two days before flowering stage (T5), the endogenous hormone contents in X-MS showed deficits and other abnormalities as the development of flower buds, the antioxidant enzyme activities showed abnormal changes, and starch and sucrose contents generally showed a shortfall trend. From T1 to T3, 1,947 differentially expressed genes were identified through transcriptome sequencing, among which 911 were up-regulated and 1036 were down-regulated. GO enrichment analysis indicated that catalytic activity, binding function, cellular process, and metabolic process were the most annotations in molecular function and biological process. KEGG annotation found that the differential genes were mainly enriched in pathways such as plant hormone signal transduction, phenylpropanoid biosynthesis, starch and sucrose metabolism, and amino sugar and nucleotide sugar metabolism. By analyzing the pathways of plant hormone signal transduction, phenylpropanoid biosynthesis, and starch and sucrose metabolism, further screening for genes related to peroxidase, sucrose synthase, and IAA. These genes may play important roles in the pollen development process of zucchini of male sterile, and provide a reference for further research on the molecular mechanism of male sterility in zucchini.