<i>Purpose:</i> Holmium-166 (¹⁶⁶Ho) is a neutron-activated radioactive isotope whose effectiveness in hepatocellular carcinoma (HCC) was first reported in a preclinical study in 1991. Chitosan is a polymer of 2-deoxy-2-amino-<i>D</i>-glucose that readily forms a chelate with heavy metals and converts from a solution under acidic conditions into a gel under neutral or basic conditions. We performed a prospective trial of a transarterial administration of a radiopharmaceutical ¹⁶⁶Ho-chitosan complex in patients with single, large HCC. <i>Patients and Methods:</i> The study involved 54 patients who had single HCC (≥3 cm) without a vascular shunt and were either inoperable or refused surgery. The ¹⁶⁶Ho-chitosan complex was administered at a dose of 20 mCi per cm of tumor diameter (capping at 200 mCi) via the artery that directly fed the tumor. <i>Results:</i> The median tumor size was 5.3 cm (range: 3–13 cm). The response rate was 78% (42/54), and 31 patients had a complete response for a median duration of 27 months. The incidence of grade 3 or 4 leukopenia was 18.6%, anemia 7.4%, thrombocytopenia 27.8%, AST/ALT elevation 26%/24%, and total bilirubin elevation 5.6%. There were two treatment-related deaths (3.7%). Subset analysis revealed a substantial difference between the two groups categorized by tumor size (3–5 vs. >5 cm) with respect to response rate (p = 0.004) and overall survival (p = 0.02). <i>Conclusion:</i> We found that transarterial administration of the ¹⁶⁶Ho-chitosan complex was highly effective in the treatment of HCC with acceptable toxicities, especially for patients with tumors of 3–5 cm.