Impaired function of the anterior cingulate cortex (ACC) is widely recognized as a critical factor in drug dependence. This study aimed to investigate protein alterations in the ACC of morphine-dependent mice using 4D label-free quantitative proteomics. Procrustes analysis and Pearson correlation analysis of the differentially expressed proteins (DEPs) and behavioral phenotypes identified 81 DEPs (p-value < 0.05). Advanced bioinformatics analysis of these DEPs suggested dysregulation of several biological pathways in the ACC of morphine-dependent mice, including mitochondrial energy metabolism, endoplasmic reticulum-to-nucleus signaling, inhibitory synapse assembly, and intracellular trafficking, secretion, and vesicle-mediated transport. These findings provide a basis for understanding the proteomic alterations and offer an integrated perspective on the biomolecular changes in the ACC associated with morphine dependence.