Diisononyl phthalate (DiNP) is a higher-molecular weight phthalate used to make plastics more flexible and durable. DiNP usage is increasing due to replacing another similar phthalate, di(2-ethylhexyl) phthalate (DEHP). However, DiNP exposure has shown negative effects on female reproductive health due to its endocrine disrupting chemical (EDCs) properties. Ingestion is the most common route of DiNP exposure, making the gastrointestinal tract and its metabolizing and hormone secreting gut-microbiome directly exposed to EDCs. The gut-microbiome has been shown to influence female reproductive health and conditions. This study is examining the relationship between acute DiNP exposure and the absence or presence of the gut microbiome on uterine development. Female C57Bl/6 germ-free (GF) one-month old mice are dosed with either sterile PBS to stay GF or dosed with colon contents to develop a gut-microbiome (DGM) for 3 days. Within the GF and DGM treatment groups, half receive corn oil while half are exposed to 200 μg DiNP/kg for 10 days. The uterus is collected and analyzed for uterine development via hematoxylin and eosin staining for uterine measurements and immunohistochemistry for analyzing uterine cell proliferation, myometrium development, and estrogen receptor expression. Uterine development was shown to be influenced by the initial absence of a gut-microbiome, but DiNP exposure had no effect on uterine development either in the absence or presence of a gut-microbiome. More research is needed to analyze how DiNP exposure, and the gut-microbiome are directly impacting uterine development.