Abstract: mTOR inhibitors are frequently used in the treatment of metastatic renal cell cancer (mRCC). mTOR regulates cell growth, proliferation, angiogenesis, and survival, and additionally plays an important role in immune regulation. Since mTOR inhibitors were shown to benefit immunosuppressive regulatory T-cell (Treg) expansion, this might suppress antitumor immune responses. Metronomic cyclophosphamide (CTX) was shown to selectively deplete Tregs. This study was, therefore, designed to determine the optimal dosage and schedule of CTX when combined with everolimus to prevent this potentially detrimental Treg expansion. In this national multi-center phase I study, patients with mRCC progressive on first line anti-angiogenic therapy received 10 mg everolimus once daily and were enrolled into cohorts with different CTX dosages and schedules. Besides immune monitoring, adverse events and survival data were monitored. 40 patients, 39 evaluable, were treated with different doses and schedules of CTX. Combined with 10 mg everolimus once daily, the optimal Treg depleting dose and schedule of CTX was 50 mg CTX once daily. 23 (59%) patients experienced one or more treatment-related ≥ grade 3 toxicity, mostly fatigue, laboratory abnormalities and pneumonitis. The majority of the patients achieved stable disease, two patients a partial response. Median PFS of all cohorts was 3.5 months. In conclusion, the optimal Treg depleting dose and schedule of CTX, when combined with everolimus, is 50 mg once daily. This combination leads to acceptable adverse events in comparison with everolimus alone. Currently, the here selected combination is being evaluated in a phase II clinical trial. Trial registration: NCT01462214.