Imidacloprid (IMI) is a neonicotinoid insecticide widely used in agriculture, which binds to the nicotinic acetylcholine receptor (nAChR). Increasing evidence shows the potential risk to humans of IMI exposure and IMI has been postulated as an endocrine disruptor. For other neonicotinoids, their ability to bind and activate the G protein-coupled estrogen receptor (GPER) was probed. Given that nAChR and GPER activation are implicated in breast cancer, we hypothesize that IMI exposure produces alterations in the mammary gland that favor tumorigenesis. Mammary epithelial cell line NMuMG was exposed to IMI (0.01-10 µM) or vehicle (DMSO) for 24 h. IMI does not alter cell viability (MTT assay) but promotes cell motility. IMI boosted cell migration at 1 and 10 µM (wound healing assay) and the activity of metalloprotease (MMP)2 at 10 µM and MMP9 at 0.1 and 10 µM (gel zymography). In addition, 10 μM IMI increased GPER and α7-nAChR protein levels after 24 h of treatment, as well as c-Src phosphorylation, a kinase downstream of GPER and α7-nAChR, after 1, 2 and 4 h (western blot). Next, cells were preincubated with specific inhibitors -1 μM G15 for GPER, 1 μM mecamylamine for nAChR, 1 μM methyllycaconitine citrate for α7-nAChR or 0.2 nM PP2 for c-Src- for 1 h and cell migration was assayed. Results showed IMI-promoted wound healing was blocked in the presence of each inhibitor. Finally, female pre-pubertal BALB-c mice were treated with IMI (0.01, 0.1 and 10 mg/kg/day) orally for 4 weeks and the whole mammary gland was mounted and hematoxylin-eosin stained sections were examined. IMI (10 mg/kg/day) enhanced ductal hyperplasia and the number of terminal end buds (TEBs). IMI (0.1 mg/kg/day)-treatment induced ductal growth but reduced branch density. In summary, IMI increases mammary epithelial cell motility through GPER and α7-nAChR and promotes preneoplastic lesions and TEBs retention. Our results support the hypothesis that IMI represents a risk factor for breast cancer.