Background: Folate receptor alpha (FRα) is overexpressed by multiple cancers, including breast and ovarian cancers. Endogenous T-cell immunity to each of five degenerate peptides from FRα (FR30, FR56, FR76, FR113 and FR238) has been demonstrated in both breast and ovarian cancer patients, suggesting the feasibility of targeting FRα via a vaccine approach. Metronomic oral cyclophosphamide (CTX) has been demonstrated to reduce immunosuppressive T regulatory cells (Tregs) and might thereby improve vaccine efficacy. We therefore conducted a Phase I clinical trial testing safety and immunogenicity of a multi-epitope FRα peptide vaccine after 1 cycle of CTX. Methods: Twenty-two patients with breast or ovarian cancer who had undergone standard surgery and adjuvant treatment were treated with 1 cycle of CTX (given days 1-7 and 15-22 of 28). Following this, patients were vaccinated intradermally at 3 sites with a mixture of the 5 FRα peptides on day 1 of a 28 day cycle for a maximum of 6 vaccination cycles. Patients were monitored for toxicity at each visit. Peripheral blood samples were collected for immune monitoring purposes at baseline, at the completion of each cycle of treatment, and at during observation at 3, 6, and 12 months after vaccine completion. The number of Tregs was assessed via flow cytometry, and the number of antigen-specific T cells was assessed via ELIspot. Results: Of 22 patients evaluable for toxicity, one patient developed a grade 3 injection site reaction. Grade 2 reactions were observed in 14 patients and included lymphopenia (5), neutropenia (4), injection site reactions (2), and leukopenia (2). The median frequency of Tregs did not significantly change after CTX use. However, FRα-specific T cell responses were observed in 20 of 21 patients with immune response data. Of these, 16 of 16 patients with observation data had demonstrable T cell responses persisting into the observation phase. Conclusions: Vaccine treatments were associated with mild to moderate toxicity. FRα specific T cell responses were induced in the majority of patients and frequently persisted after completion of vaccine therapy. Clinical trial information: NCT01606241