In mammals, the cumulus-oocyte complex (COC) is the main component of ovarian follicles. Bi-directional communication between oocytes and surrounding cumulus granulosa cells is essential for the development of oocytes and ovarian follicles. In this study, we performed proteomic profiling of mouse mature COC, using two-dimensional gel electrophoresis and mass spectrometry. A total of 259 protein spots were identified, which correspond to 156 individual proteins. We also discovered some protein families, which may play important roles in ovarian follicular development. Immunostaining was conducted to determine the subcellular localization of specific proteins from selected protein families of interest, and to examine their expression patterns during follicle development. These data provide valuable information for future studies to identify proteins involved in ovarian follicular development and related reproductive abnormalities.