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Scarce data exist regarding the incidence of donor-specific antibodies (DSAs) in kidney transplant patients receiving everolimus-based immunosuppression without calcineurin inhibitors (CNIs). The aim of this retrospective case–control study was to compare the incidence of de novoDSAs in patients converted to an everolimus-based regimen without CNIs with that seen in patients maintained on CNIs. Sixty-one DSA-free kidney transplant patients who had been converted to an everolimus-based regimen (everolimus group) were compared to 61 other patients maintained on CNIs-based regimen (control group). Patients were matched according to age, gender, induction therapy, date of transplantation, and being DSA-free at baseline. At last follow-up, the incidence of DSAs was 9.8% in the everolimus group and 5% in the control group (p = ns). In the everolimus group, the increased incidence of DSAs between baseline and last follow-up was statistically significant. Antibody-mediated rejection occurred in 6.5% in the everolimus group and 0% in the CNIs group. The incidence of DSAs is numerically increased in kidney transplant patients treated with an everolimus-based without CNIs. A study including a larger number of patients is required to determine whether a CNI-free everolimus-based immunosuppression significantly increases DSAs formation.