Key message <p>Low-temperature stress (LTS) inhibits the growth and yield of pitaya. HuBBX1, a cold-tolerance transcription factor, was identified based on the physiological and transcriptomic analyses between two pitaya cultivars. HuBBX1 enhances plant cold tolerance via the activation of cold-regulated (<i>COR</i>) genes.</p> Abstract <p>Low-temperature stress (LTS) affects fruit quality and yields. Pitaya plants are sensitive to LTS; however, the molecular mechanisms underlying LTS in pitaya remain poorly understood. In this study, the chlorophyll fluorescence, photosynthetic system, cold tolerance-associated physiological indexes, and differentially expressed genes (DEGs) were compared using two pitaya cultivars, i.e., ‘SCAU-NH’ (resistant to low temperature) and ‘SCAU-KX’ (sensitive to low temperature) at the LTS. Six candidate transcription factors (TFs) related to LTS were identified according to their expression profiles. Yeast growth assay and GUS activity analysis showed that HubZIP6, HuOFP12, HuBBX1, HuHY5, HuBOA1, and HuERF39 could enhance cold resistance. Among them, HuBBX1 is a nuclear protein with transcriptional activation capability. Overexpression of <i>HuBBX1</i> enhanced cold tolerance in <i>Arabidopsis thaliana</i> and tomato. Dual-luciferase reporter assays (DLR), yeast one hybrid (Y1H), and EMSA showed that HuBBX1 promotes the expression of cold-regulated (<i>COR</i>) genes (<i>HuCOR15A </i>and <i>HuRD29A</i>) by directly bounding to the G-box in their promoters to increase plant cold tolerance. The present work provides a foundation for further study of the molecular mechanisms of pitaya’s response to LTS.</p>