Sleep deprivation (SD) during pregnancy can impact the delivery procedure, with prolongation of the labor duration. Matrix metalloproteinase-9 (MMP9) and transforming growth factor-β (TGF-β) are regulators of uterine remodeling. Their dysregulation is vital for abnormal placentation and uterine enlargement in complicated pregnancies. Therefore, this study aims to explore the outcome of SD throughout pregnancy on ex vivo uterine contractility, MMP9 and TGF-β, and uterine microscopic structure. A total of 24 pregnant rats were divided into two groups. From the first day of pregnancy, animals were exposed to partial SD/6 h/day. Uterine in vitro contractile responses to oxytocin, acetylcholine, and nifedipine were assessed. Additionally, uterine levels of superoxide dismutase and malondialdehyde and uterine mRNA expression of MMP9, TGF-β, and apoptotic biomarkers were analyzed. The results showed that SD significantly reduced uterine contractile responses to oxytocin and acetylcholine, while it augmented the relaxing effect of nifedipine. In addition, it significantly increased oxidative stress status, MMP9, TGF- β, and apoptotic biomarkers’ mRNA expression. All were accompanied by degeneration of endometrial glands, vacuolization with apoptotic nuclei, and increased area% of collagen fibers. Finally, increased uterine MMP9 and TGF-β mRNA expression during SD clarified their potential role in modulating uterine contractility and structure.