BACKGROUND: Progesterone, recommended for preventing preterm birth (PTB) in high-risk women, is traditionally administered via oral capsules, vaginal gels, and oil injections, which pose issues like low bioavailability, systemic side effects, and irritation, leading to reduced compliance. To address these issues, a user-friendly administration approach to deliver progesterone was needed to development for the prevention of PTB. METHODS: We developed a progesterone microemulsion using ultra high-speed homogenization, optimizing formulation parameters and confirming stability. Subsequently, progesterone microemulsion-loaded microneedle (MN) patches were created, and its morphology, strength, and biocompatibility were assessed. The pharmacokinetics of these MN patches were then evaluated using LC/MS/MS. A mouse model was used to evaluate the therapeutic effects of the MN patch, with cell cytotoxicity, blood routine, and biochemistry tests assessing its biocompatibility. RESULTS: Benzyl benzoate and triglycerides were utilized as oil solvents, and Tween 80 served as the emulsifier in the preparation of a progesterone microemulsion. This formulation exhibited a particle size of 180.8 ± 20.5 nm, a zeta potential of -17.5 ± 3.4 mV, and a concentration of 20.59 ± 1.28 mg/mL. The particle size, zeta potential, and concentration of the sterilized microemulsion remained stable under 4°C. The prepared MN patch uses Povidone K30 and sucrose as excipients, which can maintain good hardness, intact needle shape, and constant drug concentration in the short term. The MN patch delivers progesterone with AUC and Cmax similar to oral progesterone microemulsion. In the preterm birth animal model, the median delivery days of mice in the progesterone microemulsion oral group and MN patch group were 19 and 20, respectively, and there was no statistical difference between the two groups. After using MN patches, the pores formed can quickly heal within 24 hours. After multiple uses of MN patches, significant abnormalities were not found in the blood routine, biochemical tests, and major organs of mice. CONCLUSION: Microneedle patches loaded with progesterone microemulsion were successfully developed, efficiently delivering progesterone and reversing RU486-induced preterm birth in mice. The MN patch was user-friendly, minimally harmful to skin tissue, safe, and non-toxic, representing a promising new approach for the clinical treatment of premature labor.