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Background  Suboptimal root zone temperature (RZT) causes a remarkable reduction in growth of horticultural crops during winter cultivation under greenhouse production. However, limited information is available on the effects of suboptimal RZT on nitrogen (N) metabolism in cucumber seedlings. The aim of this study is to investigate the effects of 24-Epibrassinolide (EBR) on nitrate and ammonium flux rate, N metabolism, and transcript levels of  NRT1  family genes under suboptimal RZT in cucumber seedlings.    Results  Suboptimal RZT (LT) negatively affected on cucumber growth and proportionately decreased EBR contents, bleeding rate, root activity, enzyme activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), and glutamate synthase (GOGAT), nitrate (NO 3  − ) influx rate, ammonium (NH 4  + ) efflux rate, and transcript levels of nitrate transporter ( NRT1 ) encoding genes. However, exogenous EBR reduced the harmful effects of suboptimal RZT and increased endogenous EBR contents, bleeding rate, root activity, enzyme activities of NR, NiR, GS, and GOGAT, NH 4  +  and NO 3  −  flux rates and contents, and N accumulation. EBR-treated seedlings also upregulated the transcript levels of nitrate transporters  CsNRT1.1 ,  CsNRT1.2A ,  CsNRT1.2B ,  CsNRT1.2C ,  CsNRT1.3 ,  CsNRT1.4A ,  CsNRT1.5B ,  CsNRT1.5C ,  CsNRT1.9 , and  CsNRT1.10 , and downregulated  CsNRT1.5A  and  CsNRT1.8 . LT treatment upregulated the expression level of  CsNRT1.5A , while exogenous BZR application downregulated the expression level of  NRT1  genes.    Conclusion  These results indicate that exogenous application of EBR alleviated the harmful effects of suboptimal RZT through changes in N metabolism, NH 4  +  and NO 3  −  flux rates, and  NRT1  gene expression, leading to improved cucumber seedlings growth. Our study provides the first evidence of the role of EBR in the response to suboptimal RZT in cucumber, and can be used to improve vegetable production.    Electronic supplementary material  The online version of this article (10.1186/s12870-019-1838-3) contains supplementary material, which is available to authorized users.

24-Epibrassinolide promotes NO3− and NH4+ ion flux rate and NRT1 gene expression in cucumber under suboptimal root zone temperature