Background: The achievement of a pathological complete response with preoperative concurrent chemoradiotherapy in locally advanced rectal cancer has been found to correlate with a reduced incidence of local and distant recurrences. With the radiosensetizing properties of gemcitabine, we tested the efficacy and toxicity of preoperative concurrent infusional gemcitabine and radiotherapy in locally advanced rectal cancer. Methods: This was a phase II, single-arm, single-institution trial. Eligibility included a diagnosis of rectal adenocarcinoma with stage T2–4 and/or nodal involvement by MRI and endoscopic rectal ultrasound, age ≥ 18 years and no prior chemotherapy or radiotherapy. Patients received preoperative radiation at a dose of 50.4–54 Gy over 28 days with concurrent infusional gemcitabine administered at a dose of 100 mg/m2 over the course of 24 hours weekly for 6 weeks. The primary endpoint was a pathological complete response (pCR). The trial was registered at clinicaltrials.gov (NCT02919878). Results: Forty patients were enrolled in the study. All of the patients completed the planned therapy, except for one patient who died at the end of his concurrent chemoradiation. Eight patients did not undergo surgery, with 1 patient dying (mentioned above), 2 patients progressing to nonresectable disease and 5 patients withdrawing consent. Six patients progressed prior to or had metastases identified at surgery, with 2 patients having unresectable metastases, 3 patients having resectable liver metastases and 1 patient having a peritoneal metastasis (not resected). Serious adverse events were reported in 8 patients (20%). The most common grade 3–4 toxicities in the preoperative period included lymphopenia (50%), neutropenia (41%), anemia (15%), diarrhea (12%), abdominal pain (12%) and proctitis (8%). Out of the 32 patients who underwent surgery, 7 patients achieved pCR at a rate of 20%. With a median follow-up of 30 months, 4 additional patients relapsed (all of these patients had distant metastases, with one subsequently having a local recurrence). The 3-year event-free and overall survival rates were 70% and 85%, respectively. Conclusions: Concurrent preoperative chemoradiotherapy using infusional gemcitabine for locally advanced rectal cancer achieved an encouraging local control. Distant metastasis remains not decreased. Further investigations of a preoperative regimen containing gemcitabine is warranted. Clinical trial information: NCT02919878.