Cotton cytoplasmic male sterility (CMS) has been extensively studied; however, information regarding its molecular mechanisms has not yet been disclosed. Therefore, to explore the molecular mechanism of pollen abortion of cotton CMS line H276A, transcript profiles of 30 mitochondrial protein-encoding genes at tetrad stage were conducted with northern blot and a differential expression gene cox3 was identified. Quantitative reverse-transcribed PCR (qRT-PCR) analysis indicated that the expression level of cox3 in the CMS line H276A was 0.39-fold compared to its maintainer line H276B. In addition, the immunoblot analysis revealed that the amount of COX3 protein was decreased to 59.38% in CMS line H276A. The 5` and 3` terminals of the transcript of cox3 in two materials were determined simultaneously with circularized RNA reverse-transcribed PCR (CR-RT-PCR). The data indicated that seven 5` end of transcript of cox3 in H276A (-451/-464/-465/-467/-471/-472/-508 respect to ATG) were identified which were different from that of H276B (-411/-412). A total of 15 single nucleotide polymorphisms (SNPs) was detected by clone sequencing analysis of upstream of cox3. To our knowledge, we are the first to comprehensively analyze the transcripts of the mitochondrial genome in the cotton CMS line and to identify the 5` and 3` terminals of the transcript of cox3 in cotton. Our data will provide a framework for a better understanding of molecular mechanisms of CMS and mitochondrial gene expression in cotton.