PURPOSE: We hypothesized that autophagy inhibition would increase response to chemotherapy in the preoperative setting for patients with pancreatic adenocarcinoma. We performed a randomized controlled trial to assess the autophagy inhibitor hydroxychloroquine in combination with gemcitabine and nab-paclitaxel. PATIENTS AND METHODS: Participants with potentially resectable tumors were randomized to two cycles of nab-paclitaxel and gemcitabine (PG) alone or with hydroxychloroquine (PGH), followed by resection. The primary endpoint was histopathologic response in the resected specimen. Secondary clinical endpoints included serum CA 19-9 biomarker response and margin negative R0 resection. Exploratory endpoints included markers of autophagy, immune infiltrate, and serum cytokines. RESULTS: Thirty-four patients in the PGH arm and 30 in the PG arm were evaluable for the primary endpoint. The PGH arm demonstrated statistically improved Evans grade histopathologic responses ( = 0.00016), compared with control. In patients with elevated CA 19-9, a return to normal was associated with improved overall and recurrence-free survival ( < 0.0001). There were no differences in serious adverse events between arms and chemotherapy dose number was equivalent. The PGH arm had greater evidence of autophagy inhibition in their resected specimens (increased SQSTM1, = 0.027, as well as increased immune cell tumor infiltration, = 0.033). Overall survival ( = 0.59) and relapse-free survival ( = 0.55) did not differ between the two arms. CONCLUSIONS: The addition of hydroxychloroquine to preoperative gemcitabine and nab-paclitaxel chemotherapy in patients with resectable pancreatic adenocarcinoma resulted in greater pathologic tumor response, improved serum biomarker response, and evidence of autophagy inhibition and immune activity.