Cytoplasmic male sterile (CMS) system has extensively been used for exploiting hybrid vigor in plant breeding programs. However, poor understanding of molecular mechanism of fertility restoration in CMS is still a major constraint for its application in many crops. Using advanced analytical approaches, we elucidated the molecular pathways regulating CMS induction and fertility restoration in cotton. Reproductive structures of a novel CMS (LD6A) and its maintainer (LD6B) line were analyzed physiological and proteomic responses. Significant differential expression of proteins, such as Abrin, malate dehydrogenase, malic enzyme, isocitrate dehydrogenase, histone acetyltransferase was observed in novel CMS and its maintainer line. Transmission electron micrographs of mitochondrial structure in anther tapetum showed that the inner ridge of mitochondria in CMS line was relatively indistinct than that of LD6B with narrower membranous space at the tetrad stage. Further, relatively higher reactive oxygen species were accumulated in the anther of CMS than its maintainer line at pollen mother cell and tetrad stage. We suggest that abnormal sequence of mitochondrial ribosome gene rps4 and rpl10 and high expression of ribosome-inactivating protein gene Abrin in CMS line damaged mitochondrial membrane and consequently induced pollen sterility. These data provide new insight into CMS mechanism in cotton crops and a tool to develop new CMS germplasm resources.