Harvested cucumber is sensitive to low temperature, which is subjected to chilling injury during cold storage and causes heavy postharvest losses. In this study, the method of RNA sequencing combined with bioinformatics analysis was used to analyze the transcriptomic changes of harvested cucumber when subjected to a short-term of cold treatment. The chilling injury index (CII) and relative electrical conductivity (REC) gradually increased but the chlorophyll fluorescence (F_v/F_m) decreased with increasing time when stored at 5℃, indicating that the chilling injury was obviously occurred. Compared with that before treatment (0 h), treatment at 5℃ for 12 h resulted in 2679 differentially expressed genes (DEGs), of which 1194 DEGs were up-regulated and 1485 DEGs were down regulated. Compared with treatment for 0 h, treatment for 72 h resulted in 9494 DEGs, of which 4807 DEGs were up-regulated and 4687 DEGs were down regulated. The results of KEGG enrichment analysis showed that the DEGs caused by cold treatment were most significantly enriched in the pathways of plant hormone signal transduction, phenylalanine metabolism, plant pathway interaction and phenylpropanoid biosynthesis. The results of GO enrichment analysis showed that DEGs were mainly involved in regulation of transcription DNA templated, protein phosphorylation and transmembrane transport in biological process. In cellular component, most enriched DEGs were related to organelle components such as membrane and nucleus. In molecular function, the enriched DEGs were mainly involved in ATP binding, protein serine/threonine kinase activity, DNA binding and transcription factor activity. DEGs related to hormone signal were significantly expressed when cucumber fruits were cold-treated for 12 h. However, the DEGs related to plant pathogen interaction and phenylpropanoid synthesis were significantly induced following 72 h of cold treatment. The results showed that plant hormone participated in the transmission of low temperature signals, the induced pathogenesis-related gene and phenylpropanoid biosynthesis might be important mechanisms for harvested cucumbers to improve chilling tolerance and cope with chilling stress. The results would provide new insights into the molecular mechanisms of harvested cucumber adaptation and resistance to chilling stress, and identify a lot of key gene resources for breeding cold-tolerant cucumber.