Purpose Cardiac resynchronization therapy (CRT) is well acknowledged as an effective treatment for dyssynchronous heart failure. However, the molecular mechanism is unclear to date. Mitochondrial dysfunction and impaired energetic metabolism are two important mechanisms that lead to heart failure. Therefore, we aim to screen the changes of mitochondria-associated proteins and signaling pathways involved in heart failure and CRT treatment. Methods A total of 24 beagle dogs were randomly assigned into control (CON), heart failure (HF), or CRT group. Myocardial mitochondria from the free wall of left ventricle was extracted for isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with two-dimensional liquid chromatography tandem mass spectrometry analysis (2DLC-MS/MS). Results A total of 2190 proteins were identified, among which 234 proteins were differentially expressed in HF compared with CON group, 151 proteins were differentially expressed in CRT compared with HF group. A total of 192 of the 234 differentially expressed proteins in HF group were changed oppositely by CRT treatment, and 128 of the 151 CRT-induced differentially expressed proteins showed opposite trend of expression to HF/CON. Gene Ontology analysis of the 128 proteins revealed that 16 were localized in mitochondria, 17 were associated with calcium signaling, and 7 could be secreted extracellularly for cell-to-cell signaling. Calpain-1 (CAPN1), which is localized to mitochondria and related to calcium signaling, was upregulated in HF and downregulated after CRT treatment. CRT treatment also improved mitochondrial morphology and function and reduced collagen areas of both interstitial and perivascular fibrosis. Conclusions CRT treatment significantly improved cardiac function, reduced myocardial fibrosis, and enhanced mitochondrial function in the failing heart through CAPN1 downregulation.