Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. The uterine proteins were separated using 2‐DE. Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty‐one spots were identified as differentially expressed proteins, of which 10 were up‐regulated proteins such as alpha‐fetoprotein, chloride intracellular channel 1, transgelin, heat‐shock protein beta‐1, and carbonic anhydrase II, while 11 were down‐regulated proteins such as X‐box binding protein, glutathione S‐transferase omega 1, olfactory receptor Olfr204, and metalloproteinase‐disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.