Improvement of low nitrogen (LN) tolerance or nitrogen use efficiency (NUE) in crops is imperative for environment-friendly agriculture development. The basic helix-loop-helix (bHLH) transcription factors are involved in multiple abiotic stresses and are suitable as candidate genes for improving LN tolerance. Few studies were performed on the characterization of theHvbHLHgene family and their function in response to LN stress in barley. In this study, 103HvbHLHgenes were identified through genome-wide analysis. HvbHLH proteins were classified into 20 subfamilies based on phylogenetic analysis in barley, which was supported by conserved motifs and gene structure analysis. The stress-relatedcis-element analysis in the promoters showed thatHvbHLHsare probably involved in multiple stress responses. By phylogenetic analysis ofHvbHLHsand bHLHs in other plants, someHvbHLHswere predicted to play roles in response to nutrition deficiency stress. Furthermore, at least 16HvbHLHswere differentially expressed in two barley genotypes differing in LN tolerance under LN stress. Finally, overexpression ofHvbHLH56enhanced LN stress tolerance in transgenic Arabidopsis, suggesting it is an important regulator in LN stress response. The differentially expressedHvbHLHsidentified herein may be valuable for the breeding of barley cultivars with LN tolerance.