Background: Rat renal transplantation is an essential experimental model for studies of transplantation immunobiology. Harvesting both kidneys from one donor rat for transplantation is widely used to reduce the number of experimental animals. Using the conventional method, both kidneys of the donor rat are harvested simultaneously, which leads to the prolonged warm ischemic times during transplantation of the second donor kidney. Prolonged warm ischemia time is the main risk factor for delayed graft function. Methods: Two different approaches are compared. Method 1, conventional method: both kidneys of the donor rat are harvested simultaneously and then transplanted into two recipients. During transplantation, the first and second donor kidneys were regarded as Group 1 and 2, respectively. Method 2, step-by-step method: after left nephrectomy, the donor rat survives, and we perform left renal transplantation (Group 3). Then, the right kidney of the surviving donor rat is incised and transplanted into the left side of the second recipient (Group 4). Results: The success rates were 86.7, 93.3, 93.3 and 86.7% in groups 1, 2, 3 and 4, respectively. The warm ischemia times increased significantly in group 2 compared with the other 3 groups (p < 0.05) but differed non-significantly between groups 3 and 4 (p > 0.05). Serum creatinine levels, blood urea nitrogen and 24-h urine protein level obviously increased after kidney transplantation in group 2 compared with other groups (p < 0.05). Conclusions: We developed an optimized method for reducing warm ischemia time, thereby minimizing delayed graft function.