Compared to regular tumor cells, cancer stem cells exhibit dangerous characteristics, including high proliferation, high metastatic potential, and significantly increased in vivo tumorigenicity. Although some studies have emphasized the impact of the microenvironment on cell stemness, they have largely overlooked the mechanical forces derived from the stiffness of the surrounding extracellular matrix. Our previous research demonstrated that a 90 Pa soft fibrin matrix in three-dimensional (3D) culture can induce cells to become cancer repopulating cells with high stemness. Acetylation modification significantly influences the metabolism, epigenetics, proliferation, migration, and immune evasion of tumor cells. In this study, we performed a comprehensive analysis of the proteome and acetyl-proteome of breast cancer cells under two-dimensional (2D) plate and 3D matrix conditions with varying stiffness. This dataset provides a valuable resource for understanding the dynamic regulation of protein acetylation in response to mechanical stiffness. The mass spectrometry-based proteomics data have been uploaded to the ProteomeXchange Consortium with the dataset identifier PXD057820.