BACKGROUND AND AIMS: Among the greatest hurdles to pancreatic cancer (PC) therapy is the limited tissue penetration of systemic chemotherapy because of tumor desmoplasia. The primary study aim was to determine the toxicity profile of EUS-guided fine-needle injection (EUS-FNI) with gemcitabine. Secondary endpoints included the ability to disease downstage leading to an R0 resection and overall survival (OS) at 6 months, 12 months, and 5 years after therapy. METHODS: In a prospective study from a tertiary referral center, gemcitabine (38 mg/mL) EUS-FNI was performed in patients with PC before conventional therapy. Initial and delayed adverse events (AEs) were assessed within 72 hours and 4 to 14 days after EUS-FNI, respectively. Patients were followed for ≥5 years or until death. RESULTS: Thirty-six patients with stage II (n = 3), stage III (n = 20), or stage IV (n = 13) disease underwent gemcitabine EUS-FNI with 2.5 mL (.7-7.0 mg) total volume of injectate per patient. There were no initial or delayed AEs reported. Thirty-five patients (97.2%) were deceased at the time of analysis with a median 10.3 months of follow-up (range, 3.1-63.9). OS at 6 months and 12 months was 78% and 44%, respectively. The median OS was 10.4 months (range, 2.7-68). Among patients with stage III unresectable disease, 4 (20%) were downstaged and underwent an R0 resection. CONCLUSIONS: Our study suggests the feasibility, safety, and potential efficacy of gemcitabine EUS-FNI for PC. Additional data are needed to verify these observations and to determine the potential role relative to conventional multimodality therapy.