AbstractCold stress affects the growth and development of cucumbers. Whether the BPC2 transcription factor participates in cold tolerance and its regulatory mechanism in plants have not been reported. Here, we used wild-type (WT) cucumber seedlings and two mutant Csbpc2 lines as materials. The underlying mechanisms were studied by determining the phenotype, physiological and biochemical indicators, and transcriptome after cold stress. The results showed that CsBPC2 knockout reduced cucumber cold tolerance by increasing the chilling injury index, relative electrical conductivity and malondialdehyde (MDA) content and decreasing antioxidant enzyme activity. We then conducted RNA sequencing (RNA-seq) to explore transcript-level changes in Csbpc2 mutants. A large number of differentially expressed genes (1032) were identified and found to be unique in Csbpc2 mutants. However, only 489 down-regulated genes related to the synthesis and transport of amino acids and vitamins were found to be enriched through GO analysis. Moreover, both RNA-seq and qPT-PCR techniques revealed that CsBPC2 knockout also decreased the expression of some key cold-responsive genes, such as CsICE1, CsCOR413IM2, CsBZR1 and CsBZR2. These results strongly suggested that CsBPC2 knockout not only affected cold function genes but also decreased the levels of some key metabolites under cold stress. In conclusion, this study reveals for the first time that CsBPC2 is essential for cold tolerance in cucumber and provides a reference for research on the biological function of BPC2 in other plants.