Classical immunosuppression based on steroids, calcineurin inhibitors, and mycophenolate results in several unwanted effects and unsatisfactory long-term outcomes in kidney transplantation (KT). New immunosuppressors search for fewer adverse events and increased graft survival but may have a distinct impact on graft function and immunological biomarkers according to their mechanism of action. This prospective study evaluates the immunological effect of tacrolimus to serine/threonine protein kinase mechanistic target of rapamycin inhibitors (mTORi) conversion in 29 KT recipients compared with 16 controls maintained on tacrolimus. We evaluated renal function, human leukocyte antigen (HLA) antibodies and peripheral blood lymphocyte subsets at inclusion and at 3, 12, and 24 months later. Twenty immunophenotyped healthy subjects served as reference. Renal function remained stable in both groups with no significant change in proteinuria. Two patients in the mTORi group developed HLA donor-specific antibodies and none in the control group (7% vs. 0%, p = 0.53). Both groups showed a progressive increase in regulatory T cells, more prominent in patients converted to mTORi within the first 18 months post-KT (p < 0.001). All patients showed a decrease in naïve B cells (p < 0.001), excepting those converted to mTORi without receiving steroids (p = 0.31). Transitional B cells significantly decreased in mTORi patients (p < 0.001), independently of concomitant steroid treatment. Finally, CD56bright and CD94/NK group 2 member A receptor positive (NKG2A+) Natural Killer (NK) cell subsets increased in mTORi- compared to tacrolimus-treated patients (both p < 0.001). Patients switched to mTORi displayed a significant redistribution of peripheral blood lymphocyte subpopulations proposed to be associated with graft outcomes. The administration of steroids modified some of these changes.