To explore the effects of the ASB14 gene on changes in mitochondria-associated protein levels and signaling pathways, a proteomic analysis containing blank cell control, empty vector control, and AC16 stably transfected cell strain overexpressing the ASB14 was included in the subproject. By comparing protein expression profiles among AC16 cells transfected with pCDH-NC and pCDH-ASB14, a total of 1306 differential proteins were obtained in this study, of which 495 were up-regulated and 811 were down-regulated. These proteins were significantly enriched into 32 KEGG pathways, including linoleic acid metabolism. ASB14 overexpression may mediate the metabolic level of linoleic acid by regulating differential proteins and further affects mitochondrial OXPHOS activity in cardiomyocytes.