Therapeutic drug monitoring (TDM) for tacrolimus (Tac) is universally applied. However, the concentration–effect relationship for Tac is poorly defined. This study investigated whether Tac concentrations are associated with acute rejection in kidney transplant recipients. Data from three large trials were pooled. We used univariate and multivariate analysis to investigate the relationship between biopsy-proven acute rejection (BPAR) and Tac predose concentration at five time points (day 3, 10 and 14, and month 1 and 6 after transplantation). A total of 136/1304 patients experienced BPAR, giving an overall incidence of 10.4%. We did not find any significant correlations between Tac predose concentrations and the incidence of BPAR at the different time points. In the multivariate analysis, only delayed graft function (DGF) and the use of induction therapy were independently correlated with BPAR, with an odds ratio of 2.7 [95% CI: 1.8–4.0; p < 0.001] for DGF and 0.66 [95% CI: 0.44–0.99; p = 0.049] for induction therapy. The other variables, including the Tac predose concentrations, were not statistically significantly associated with BPAR. We did not find an association between the Tac predose concentrations measured at five time points after kidney transplantation and the incidence of acute rejection occurring thereafter. Based on this study it is not possible to define the optimal target concentrations for Tac.