We set out to determine the effects of mtDNA haplotype on the developmental potential of cattle oocytes using somatic cell nuclear transfer (SCNT) to generate embryos. To ensure that only one mtDNA haplotype was transmitted, mtDNA was depleted from donor fibroblasts prior SCNT by treatment with 2’, 3’-dideoxycytidine. SCNT embryos were produced using Bos taurus (Holstein) donor fibroblasts and recipient oocytes from Bos taurus (Angus) and Bos indicus (Sahiwal) cattle. Firstly, donor cells from depleted and non-depleted (control) cells were fused to enucleated Bos taurus recipient oocytes. After chemical activation, embryos were in vitro cultured for 7 days until they reached the blastocyst stage. We found that blastocysts derived from depleted cells harboured only oocyte mtDNA whilst the co-existence of donor cell and oocyte mtDNA was found in blastocysts derived from non-depleted cells. Therefore, SCNT embryos produced from depleted cells possessed only one mtDNA population. Moreover, mtDNA copy number in blastocysts derived from depleted cells was not significantly different when compared with blastocysts from non-depleted cells. Consequently, depleting mtDNA from donor cells had no negative effect on mtDNA copy number. Secondly, we generated embryos using Holstein depleted cells and the more genetically divergent Bos indicus oocytes. Sahiwal cattle were assigned to one of 2 sub-haplotypes (A and B) based on their mtDNA sequences. Blastocyst rates for embryos derived from Bos indicus oocytes (3.5%) were significantly lower than those from Bos taurus oocytes (35.0%). Interestingly, only embryos derived from group B developed to the blastocyst stage and produced one live offspring, which inherited only oocyte mtDNA. MtDNA copy number for Sahiwal oocytes from group A was significantly lower than for Bos taurus oocytes, which likely accounted for their developmental failure. In conclusion, oocytes with different mtDNA backgrounds exhibited different developmental potentials for the generation of SCNT embryos.