Plant cytoplasmic male sterility (CMS) is a maternal genetic trait that results from incompatibilities between the organellar and nuclear genomes which causes pollen abortion. Cytoplasmic male sterility plays an important role in hybrid seed production and heterosis utilization. In kenaf, cytoplasmic male sterility has been exploited in hybrid seed production for several years, but its molecular mechanism has not yet been disclosed. In the present work, the mitochondrial genome of the isonuclear cytoplasmic male sterile line UG93A compared with maintainer line UG93B was described and a total of 398 single nucleotide polymorphisms (SNPs) and 230 insertions/deletions (InDels) were identified in the UG93A mitochondrial DNA. A total of 26 single nucleotide polymorphisms and insertions/deletions were identified in the coding region of atp6, indicating active mitochondrial genome rearrangement. An expression cassette containing atp6 of UG93A without a fused mitochondrial transit peptide induced male sterility in tobacco and Arabidopsis. This indicated abnormal pollen grains and lack of pollen activity. Conclusively, coding sequence of atp6, which contains single nucleotide polymorphism and indel variations may play a crucial role in regulating male sterility in kenaf. These data suggested that atp6 of UG93A may be a candidate gene for kenaf cytoplasmic male sterility and provided basic information for further study of the molecular mechanisms of cytoplasmic male sterility in kenaf.