With an aim to improve the efficiency of yak–bovine interspecies somatic cell nuclear transfer (iSCNT), this study investigated the effect of different culture systems on the development, quality and gene expression profile of yak–bovine iSCNT embryo. Reconstructed embryos were cultured in modified synthetic oviductal fluid (mSOF) or relative defined culture medium (RDCM) with 5% or 20% oxygen tension. Relative mRNA abundance of Oct-4, IFNT, IGF-2, Bax, GPX-1, SOD-1, CAT and GSS was analysed in blastocysts with qRT-PCR. The blastocyst formation rate in RDCM under 5% oxygen tension was significantly higher than that under 20% oxygen tension (< 0.05). The total cell number of blastocyst derived from RDCM with 20% oxygen tension was lower than that of other groups, whereas the group of RDCM with 5% oxygen tension showed a beneficial effect on apoptosis index and tolerance to cryopreservation (< 0.05). However, under the same oxygen tension, the mRNA abundance of IFNT of RDCM groups was higher than that of the mSOF groups. In addition, high oxygen tension during in vitro culture (IVC) with RDCM significantly increases the mRNA expression of oxidative stress-related genes (GPX-1, SOD-1, CAT and GSS) (< 0.05). 3, 4-Dihydroxyflavone (DHF) during high oxygen tension was able to improve the cloned blastocyst formation rate in RDCM (< 0.05). These results for the first time showed that low oxygen tension and RDCM could improve the developmental competence and quality and alleviate the oxidative stress for yak–bovine iSCNT embryo during IVC.