Estrogen Receptor subtypes (ERα and ERβ) are transcription factors sharing similar structure, however, they often perform opposite roles in breast cancer’s cell proliferation and tumor progression. Besides the well-characterized genomic actions of ERs upon ligand binding, rapid non-genomic cytoplasmic changes together with the recently discovered ligand-free action of ERs are emerging as key regulators of tumorigenesis. The identification of cytoplasmic interaction partners of unliganded ERα and ERβ may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, in this study, cytoplasmic extracts from stably expressing TAP-ERα and -ERβ MCF-7 cell clones were subjected to interaction proteomics in the absence of estrogen stimulation, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERβ, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways associated to each receptor in the cytoplasm. This work reports the first identification of the unliganded ERα and ERβ cytoplasmic interactomes in breast cancer cells, providing novel experimental evidence on the non-genomic effects of ERs in the absence of hormonal stimulus.