The ubiquitin-proteasome system and the autophagy-lysosome pathway are the two main mechanisms for eukaryotic intracellular protein degradation. Proteasome inhibitors are used for the treatment of some types of cancer, whereas autophagy seems to have a dual role in tumor cell survival and death. However, the relationship between both pathways has not been extensively studied in tumor cells. We have investigated both proteolytic systems in the human epithelial breast non-tumor cell line MCF10A and in the human epithelial breast tumor cell line MCF7. In basal condition, tumor cells showed a lower proteasome function but a higher autophagy activity when compared with MCF10A cells. Importantly, proteasome inhibition (PI) leads to different responses in both cell types. Tumor cells showed a dose-dependent glycogen synthase kinase-3 (GSK-3)β inhibition, a huge increase in the expression of the transcription factor CHOP and an active processing of caspase-8. By contrast, MCF10A cells fully activated GSK-3β and showed a lower expression of both CHOP and processed caspase-8. These molecular differences were reflected in a dose-dependent autophagy activation and cell death in tumor cells, while non-tumor cells exhibited the formation of inclusion bodies and a decrease in the cell death rate. Importantly, the behavior of the MCF7 cells can be reproduced in MCF10A cells when GSK-3β and the proteasome were simultaneously inhibited. Under this situation, MCF10A cells strongly activated autophagy, showing minimal inclusion bodies, increased CHOP expression and cell death rate. These findings support GSK-3β signaling as a key mechanism in regulating autophagy activation or inclusion formation in human tumor or non-tumor breast cells, respectively, which may shed new light on breast cancer control.