Objective The Sprague-Dawley rat strain is one of most popular animal models for studies of folliculogenesis, oocyte maturation and reproductive toxicology. The aim of the present study was to determine the optimal conditions for in vitro fertilization (IVF) and culture system of Sprague-Dawley rat oocytes. Methods Rat oocytes obtained from superovulation or natural ovulation cycle were fertilized and cultured in the HTF and IVF-30 media, modified by supplement with NaCl in different concentrations. Penetration of sperm, pronuclear and blastocyst formation were observed. Results Oocytes fertilized and subsequently cultured in IVF-30 or HTFplus supplemented with 30 mmol/L NaCl showed fertilization rates (67%, 70%) with subsequent blastocyst formation rates, 22% and 32%, respectively, significantly higher than the fertilization rates (15% and 13%, respectively) with no blastocyst formation in original media. More oocytes from natural ovulation cycles (28%) developed into blastocyst than that from superovulation (16%). Conclusion An in vitro fertilization and culture system for SD rat oocytes has been successfully established.