In order to decipher the potential function of barley salt-tolerant related gene HvMBF1c and its expression pattern under salt stress treatment condition, isolation of the HvMBF1c gene via homologous cloning method was performed in two barley germplasm resources ZY26 (salt-tolerant type) and ZY218 (salt-sensitive type), followed by bioinformatics analysis of the structure, physical and chemical properties, homologous protein differences and phylogeny. HvMBF1c contains a 471bp open reading frame found on barley chromosome 7HL without intron structure, and encodes 156 amino acids. The deduced protein contains an N-terminal MBF1 domain and a C-terminal HTH domain, with the characteristics of unstable, alkaline, hydrophilic, non-transmembrane and no signal peptide structure, and its secondary structure of α helix. The phylogenetic analysis showed that HvMBF1c was highly homologous to MBF1 of various plants, and was closely related to TaMBF1c of wheat. Subcellular localization indicated that the fusion protein was mainly located in the nucleus and cytoplasm. Under NaCl stress treatment condition, the induction on the expression of the HvMBF1c gene was observed in seeds, leaves and roots at seedling stage, while the expression level in leaves was significantly higher than that in roots. The overall expression level was correlating with the ability of barley germplasm salt tolerance and the time for stress treatment. Collectively, these results demonstrated that HvMBF1c was involved in regulating the response of barley to salt stress at different growth stages.