Di-isononyl phthalate (DiNP), a common plasticizer used in polyvinyl chloride products, exhibits endocrine-disrupting capabilities. It is also toxic to the brain, reproductive system, liver, and kidney. However, little is known about how DiNP impacts the gastrointestinal tract (GIT). It is crucial to understand how DiNP exposure affects the GIT because humans are primarily exposed to DiNP through the GIT. Thus, this study tested the hypothesis that subacute exposure to DiNP dysregulates cellular, endocrine, and immunological aspects in the colon of adult female mice. To test this hypothesis, adult female mice were dosed with vehicle control or DiNP doses ranging from 0.02 to 200 mg/kg for 10-14 days. After the treatment period, mice were euthanized during diestrus, and colon tissue samples were subjected to morphological, biochemical, and hormone assays. DiNP exposure significantly increased histological damage in the colon compared to control. Exposure to DiNP also significantly decreased sICAM-1 levels, increased Tnf expression, decreased a cell cycle regulator (Ccnb1), and increased apoptotic factors (Aifm1 and Bcl2l10) in the colon compared to control. Colon-extracted lipids revealed that DiNP exposure significantly decreased estradiol levels compared to control. Collectively, these data indicate that subacute exposure to DiNP alters colon morphology and physiology in adult female mice.