Ascorbic acid (AsA) is one of the most abundant natural antioxidants, and it is an important indicator of the nutritional value of cucumber fruit. The aim of this study was to elucidate the regulatory mechanism affecting AsA metabolism in cucumber fruit. In this study, the AsA content in the fruit of two cucumber cultivars (H28 and H105) was significantly higher in the exocarp and endocarp than in the mesocarp. To clarify the regulation of AsA in cucumber fruit, the transcriptomes of three fruit tissues (i.e., the exocarp, mesocarp, and endocarp) of two cucumber cultivars (H28 and H105) were sequenced. Transcriptomic profiling combined with transcription factors (TFs) and correlation analysis were performed to reveal that three genes, including CsaV3_5G014110 (phosphomannomutase, PMM), CsaV3_2G004170 (GDP-mannose-3', 5'-epimerase, GME) and CsaV3_5G006680 (dehydroascorbate reductase, DHAR), were expressed at higher level in the exocarp and endocarp than in the mesocarp. In both two cultivars, CsaV3_4G028360 (ethylene-responsive transcription factor, ERF) was negatively correlated with PMM and GME, and positively correlated with DHAR. CsaV3_6G042110 (ethylene-responsive transcription factor, ERF) was positively correlated with PMM and GME, and negatively correlated with DHAR. CsaV3_6G032360 (mitogen-activated protein kinase, MAPK) as positively correlated with PMM, GME and DHAR. These six genes are considered the key candidate genes for further research. This study provides insight for further study on the regulation of AsA biosynthesis in cucumber fruit and provide potential candidate genes for future genetic improvement of cucumber germplasm with enhanced AsA accumulation.