The objective of this study was to characterize the pharmacokinetics and immunosuppression of a tacrolimus-loaded biodegradable microsphere (TLBM) in rats. We prepared TLBM. DA/Slc rats were given TLBM at a dose of 1.6 mg/kg (n = 9), 2.4 mg/kg (n = 5), or 7.2 mg/kg (n = 7) tacrolimus contents by a single subcutaneous administration to achieve sustained release over a long period. DA/Slc rats were given TLBM by a single subcutaneous administration at a dose of 4.8 mg/kg (n = 6) tacrolimus contents to clarify the main site of TLBM uptake in the organs. In the rat liver transplantation model, the recipients were given TLBM at a dose of 0.16 mg/kg (n = 5), 2.4 mg/kg (n = 4), or 4.8 mg/kg (n = 5) tacrolimus contents by a single subcutaneous administration on the first day after operation. Overall survival days were compared. Continuous flat parallel concentration profiles were significant for 10 days from the first day after a single subcutaneous administration of TLBM (P <.01). The relationship between the dosages of TLBM administration and area under the concentration-time curve (AUC) up to 18 days demonstrated a linear regression line (P <.01). In addition, the relationship between the dose of TLBM and the survival days of the recipients in the liver transplantation model showed a positive correlation. The current pharmacokinetic study of TLBM revealed significantly increased tacrolimus levels in the regional lymph nodes compared with other organs except bone marrow (P <.01). In conclusion, TLBM allowed tacrolimus to release gradually in a very stable manner for 10 days, with dose-dependent immunosuppression after a single subcutaneous administration. The main site of TLBM uptake after subcutaneous administration was the regional lymph node of administration site.