In breast cancer cells, 17-β-estradiol (E2) upregulates the expression of insulin receptor substrate 1 (IRS-1), a molecule transmitting insulin-like growth factor-I (IGF-I) signals through the PI-3K/Akt survival pathways. The stimulation of IRS-1 by E2 has been documented on the transcriptional level. Here we studied whether the expression of estrogen receptor (ER)-α affects IRS molecules post-transcriptionally. We used ER-α-negative MDA-MB-231 breast cancer cells and MDA-MB-231 cells with re-expressed ER-α. In MDA-MB-231 cells cultured under serum-free conditions, IRS-1 and IRS-2 were degraded through the 26S proteasome and calpain pathways. Re-expression of ER-α in MDA-MB-231 cells correlated with enhanced stability of IRS molecules. This effect coincided with significantly reduced ubiquitination of IRS-1 and IRS-2, but did not involve increased IRS-1 and IRS-2 transcription. The interference of ER-α with IRS-1 and IRS-2 turnover could rely on the competition for common degradation pathways, as in MDA-MB-231/ER cells, ER-α processing was blocked by proteasome and calpain inhibitors. Notably, a fraction of the cytosolic ER-α colocalized and coprecipitated with IRS-1 and IRS-2, indicating a possible common destination for these proteins. The stabilization of IRS-1 in MDA-MB-231/ER cells was paralleled by the upregulation of the IRS-1/Akt/GSK-3 pathway and improved survival in the presence of IGF-I, whereas IRS-2 was not involved in IGF-I signaling.