Background: Colorectal cancer (CRC) cells can become resistant to chemotherapy and anti-angiogenic therapy through autophagy. The antimalarial agent hydroxychloroquine (HCQ) is a potent inhibitor of autophagy, and in vivo studies in CRC cell models show significant decrease tumor volume when these autophagy inhibitors were combined with oxaliplatin and bevacizumab. We previously reported safety with HCQ 600mg BID in combination with standard front-line FOLFOX and bevacizumab in a Phase I study in metastatic CRC patients. We report the results of a single-arm phase II trial of patients with previously untreated stage IV CRC with good performance status and adequate hematologic and biochemical indices. Methods: Patients were treated with standard doses of mFOLFOX6 and bevacizumab with HCQ 600mg BID. Imaging was obtained every 2 months. Results: 37 patients were enrolled, 62% male, 89% Caucasian, median age 61, 65% ECOG PS 0, and 73% colon primary. Twenty-eight patients were evaluable for response as 2 patients did not start therapy and 7 patients withdrew prior to first response assessment. The ORR was 68% with an 11% CR rate. The median time to response was 3.1 months. Responses were independent of genomic aberrations within tumor tissue, specifically KRAS, TP53, BRAF, and PIK3CA. Median PFS and duration of response were not interpretable given that only 25% of patients came off trial for progression; all other patients withdrew to pursue surgery or liver embolization therapy, to receive therapy elsewhere, or due to toxicity associated with chemotherapy. The 1 year OS rate was 74%, and median OS has not been reached. The most common G3 or higher adverse events included neutropenia (31%), fatigue (11%), thromboembolism (9%), and cardiac events (9%). HCQ-attributable side effects included G1-3 insomnia (26%), G1-3 anxiety (20%), G1 visual disturbances (11%) and G3 allergy (3%). The majority of patients had increase in both LC3 and p62 in peripheral blood mononuclear cells and exhibited an increase in autophagosomes within the cytosol by electron microscopy. Conclusions: These data are promising and further evaluation in a randomized controlled trial is planned. Clinical trial information: NCT01206530