The incidence of breast cancer is increasing globally and exposure to endocrine disruptors (EDs) has become a potential risk factor for this disease. Different studies have linked breast cancer progression with pesticide exposure. Imidacloprid (IMI) is a neonicotinoid insecticide widely used and is postulated to act as an ED. Given that IMI modulates estradiol secretion in breast cancer cells, it has been suggested that it could promote this disease. Our objective was to evaluate the possible impact of IMI exposure on cell viability, proliferation, motility and estrogen receptor (ER)-α and G protein-coupled ER (GPER) protein expression in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cells. Cells were exposed to environmentally relevant concentrations of IMI (0.01-10 µM) for 24 h or vehicle (DMSO). Results showed that IMI does not alter cell viability (MTT assay), but produces an increase in cell proliferation only in MCF-7. The clonogenic assay showed an enhancement in the number of colonies at 0.01 μM, while the expression of the proliferation marker PCNA (western blot, WB) was increased at 0.1, 1 and 10 μM in MCF-7. No alterations were observed in the number of colonies of MDA-MB-231 after IMI treatment. On the other hand, WB results showed a reduction in the levels of ER-α at 0.01 μM and GPER at 10 μM in MCF-7. In addition, IMI decreased GPER expression in MDA-MB-231 at 0.01, 0.1 and 1 μM. Finally, metalloprotease-9 activity was increased in MCF-7 at all assayed doses (gel zymography), whereas cell migration was enhanced in MDA-MB-231 at 0.1 μM (wound healing assay). In conclusion, IMI exposure induces alterations in breast cancer cells that promote cell proliferation and motility, suggesting that it may be involved in breast cancer progression.