Endocrine disruptors are ubiquitous in nature. Indeed, some pesticides are not only insect killers, they also function as “endocrine disruptors” in humans and animals; therefore, concern regarding the possible health risks of pesticides for humans is growing. However, few studies have investigated the adverse effects induced by pesticides. One study previously suggested that fipronil reduced the levels of 17β-estradiol (E2), a natural ligand for estrogen receptor α (ESR1, ERα), in female Wistar rat plasma. In the present study, we focused on three relatively novel insecticides: fipronil (a phenyl pyrazole), acetamiprid (a neonicotinoid), and imidacloprid (a neonicotinoid). The effects of these 3 insecticides on the expression of ERα as well as E2/ERα-mediated signaling were examined in an ERα-positive MCF-7 human breast cancer cell line. The results obtained showed that fipronil selectively down-regulated the expression of ERα, and its regulated gene, CDC2, and also that PES1, an upstream signaling molecule for the regulation of ERα, was suppressed by the insecticide. We discussed the potential of fipronil as an antiestrogen.