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The jacalin-related lectins (JRLs) are widely distributed in plants and are involved in plant development and multiple stress responses. However, the characteristics of the <i>HvJRL</i> gene family at the genome-wide level and the roles of JRLs in barley’s response to low-nitrogen (LN) stress have been rarely reported. In this study, 32 <i>HvJRL</i> genes were identified and unevenly distributed at both ends of the seven chromosomes in barley. HvJRL proteins generally exhibited low sequence similarity but shared conserved jacalin domains by multiple sequence analysis. These proteins were classified into seven subfamilies based on phylogenetic analysis, with a similar gene structure and conserved motifs in the same subfamily. The <i>HvJRL</i> promoters contained a large number of diverse <i>cis</i>-elements associated with hormonal response and stress regulation. Based on the phylogenetic relationships and functionally known JRL homologs, it was predicted that some <i>HvJRLs</i> have the potential to serve functions in multiple stress responses but not nutrition deficiency stress. Subsequently, nine differentially expressed genes (DEGs) encoding eight HvJRL proteins were identified in two barley genotypes with different LN tolerance by transcriptome analysis. Furthermore, <i>35S:HvHorcH</i> transgenic Arabidopsis seedlings did enhance LN tolerance, which indicated that <i>HvHorcH</i> may be an important regulator of LN stress response (LNSR). The <i>HvJRL</i> DEGs identified herein could provide new candidate genes for LN tolerance studies.