Neonicotinoids are the most widely used insecticides in the world because they broadly target chewing and sucking insects. Imidacloprid (IMI) is a common neonicotinoid that accounts for 30% of neonicotinoid sales globally. IMI is used in commercial agricultural systems, sold for use in home gardens, and found in veterinary pharmaceuticals in the form of flea and tick preventatives for companion animals. IMI is also used as crop seed treatments and spreads throughout crops as they mature. Thus, IMI cannot be washed or peeled off produce. As a result, humans are routinely exposed to IMI through consumption of contaminated food and water as well as interactions with their pets. IMI kills insects by acting as a systemic neurotoxicant after binding nicotinic acetylcholine receptors (nAChRs) in the nervous system. To date, exposure to IMI has not been considered a public health concern because it is a weak agonist for mammalian nAChRs compared to insect nAChRs. However, our preliminary data indicate that the ovary bioactivates IMI to desnitro-imidacloprid (DNI). This is of concern because DNI has a higher affinity for nAChRs than IMI. Further, our preliminary data indicate that the ovary contains nAChRs and that DNI is toxic to antral follicles. Specifically, DNI exposure causes slow antral follicle growth, decreased estradiol production, follicle rupture, and increased expression of the pro-apoptotic factor Bax in mouse antral follicles in vitro. Slow follicle growth, increased Bax, follicle rupture, and low estradiol levels are of concern because they can lead to subfertility/infertility. Although IMI exposure leads to production of DNI by ovarian follicles, DNI is an ovarian toxicant in vitro, and ovarian toxicants often cause subfertility/infertility, we do not know if IMI exposure causes ovarian toxicity and female subfertility/infertility in vivo. Thus, the goal of the proposed R21 studies is to expand our preliminary data using a mouse model to test the hypothesis that IMI exposure leads to molecular changes in the ovary to cause ovarian toxicity, leading to female subfertility/infertility. To test his hypothesis, we will: 1) determine the extent to which IMI causes ovarian toxicity and female subfertility/infertility in vivo and 2) identify IMI- induced changes in molecular factors in the ovary using spatial transcriptomics. Collectively, the proposed R21 studies will determine whether IMI poses a female reproductive health hazard in mammals and should be considered for regulation of use in adult women.