Nicotinic acetylcholine receptors (nAChRs) are membrane receptors and play a major role in tumorigenesis and cancer progression. Here, we have investigated the differential expression of nAChR subunits in human breast cancer cell lines and breast epithelial cell lines at mRNA and protein levels and the effects of the αO-conotoxin GeXIVA, antagonist of α9α10 nAChR, on human breast cancer cells. Reverse transcription polymerase chain reaction (PCR) demonstrated that all nAChR subunits, except α6, were expressed in the 20 tested cell lines. Real time quantitative PCR (QRT-PCR) suggested that the mRNA of α5, α7, α9 and β4 nAChR subunits were overexpressed in all the breast cancer cell lines compared with the normal epithelial cell line HS578BST. α9 nAChR was highly expressed in almost all the breast cancer cell lines in comparison to normal cells. The different expression is prominent (p < 0.001) as determined by flow cytometry and Western blotting, except for MDA-MB-453 and HCC1395 cell lines. αO-conotoxin GeXIVA that targeted α9α10 nAChR were able to significantly inhibit breast cancer cell proliferation in vitro and merits further investigation as potential agents for targeted therapy. and non-small-cell lung carcinomas (NSCLCs), head, neck, gastric, pancreatic, gallbladder, liver, colon, breast, cervical, urinary bladder and kidney cancers [8][9][10][11][12]. Several nAChRs subunits, including α3, α7, α9 and β4, are extensively expressed in various tumor cells and they are involved in regulation of cell proliferation, apoptosis, invasion, migration and angiogenesis [13][14][15][16]. For instance, previous research showed that suppression of α7-nAChRs by snake α-neurotoxins and curare reduced the growth of NSCLC tumor [17,18], Bychkov et al. also confirmed that Ly6/uPAR proteins like Lynx1 retard the cells in G0/1 and G2/M period, inhibit the proliferation and enhance the apoptosis by down-regulating the expression of α7-nAChRs in A549 cells [8]. Furthermore, α-conotoxin AuIB could inhibit small-cell lung carcinomas (SCLC) cell viability via α3/α5/β4-containing nAChRs [12]. As for α9 nAChRs, it is important for promoting cancer cell proliferation, angiogenesis, cancer metastasis and apoptosis suppression during carcinogenesis in response to tumor microenvironments [9,19,20]. So, nAChR antagonists could potentially be used directly or in combination with established chemotherapeutic drugs to treat related cancers.As competitive antagonists of nAChRs, α-conotoxins (α-Ctxs), have been particularly useful in developing ligands that selectively target one nAChR subtype over another. Some of them have therapeutic potential [21][22][23][24][25]. αO-conotoxin GeXIVA was isolated from Conus generalis, which potently blocks α9α10 nAChR [26]. In vivo, it also displayed potent alleviation for neuropathic pain in one rat model [27,28]. Recently, our research also showed that GeXIVA contributed to the inhibition of cervical cancer cell proliferation [29]. αO-conotoxin GeXIVA have the potential for anticancer treatment both by t...