Cytochrome P450 monooxygenase (CYP450) is the largest enzyme family involved in plant metabolism, among which CYP734A subfamily members are widely involved in the inactivation of plant hormone Brassinosteroids (BRs). In order to analyze the effects of BRs on Hordeum vulgare L. var. nudum Hook. f.(highland barley), which is a characteristic plant in QinghaiTibet plateau, we treated the seedlings of highland barley cultivar ‘Dulihuang’ with the synthetic hormone 24epibrassinolide (24eBL) or BRs synthetic inhibitor brassinazole (BRZ). In order to deeply analyze the function of HvBAS1, we cloned the cDNAs from highland barley by RTPCR, and detected their expression characteristics by realtime quantitative PCR. The results showed that: (1) 24eBL could significantly promote the growth of highland barley seedlings, while BRZ was opposite. (2) Two subfamily members of CYP734As were cloned from highland barley. Due to their high homology with Arabidopsis BAS1, they were named HvBAS11 and HvBAS12. HvBAS11 was 1 629bp in length and encoded 542 aa, while HvBAS12 was 1 689 bp in length and encoded 562 aa. The homology of amino acid sequences between them was 76.42%. The results of subcellular localization predicted that both of them existed in endoplasmic reticulum. (3) Realtime quantitative PCR detection showed that the expression patterns of HvBAS11 and HvBAS12 were completely different, in which the expression of HvBAS11 in the roots was higher than that in the leaves, while the expression of HvBAS12 in the leaves was higher than that in the roots. With the increase of seedling age, the expression of HvBAS11 in the roots showed a trend of first increasing and then decreasing, but HvBAS12 decreased firstly and then increased in leaves. After BRZ treatment, both HvBAS11 and HvBAS12 were significantly downregulated compared with the control, and the expression level of HvBAS12 were decreased even more. The results indicated that both HvBAS11 and HvBAS12 were probably involved in inactivation of endogenous BRs in highland barley, but their functions were different.