In this study, we investigated the effect of ¹³¹I gelatin microspheres (¹³¹I-GMSs) on human hepatocellular carcinoma cells (HepG2) in nude mice (Balb/c) and the biodistribution of ¹³¹I-GMSs after intratumoral injection. The treatment group and control group animals received intratumoral injections of 1 mCi ¹³¹I-GMSs and GMSs unlabeled ¹³¹I, respectively. The size of the implanted tumor was measured once a week for 8 weeks, and the survival time was calculated from the day of injection to 64 days post-injection. Another 35 animals received intratumoral injections of 0.2 mCi ¹³¹I-GMSs and were subject to single-photon emission computed tomography (SPECT) on days 1, 8, 16, 24 and 32 post-injection. Samples of various organs were collected and used to calculate tissue concentrations on days 1, 4, 8, 16 and 24. Free thyroxine (FT4) in fetal bovine serum was tested to evaluate thyroid function. The tumors were collected for histological examination. ¹³¹I-GMSs produced a pronounced reduction in HepG2 tumor volume, and the overall survival was 73.3% in the treatment group and only 13.3% in the control group (P < 0.001). Tissue radioactivity concentration measurements and SPECT demonstrated that the injected ¹³¹I-GMSs mainly accumulated within the tumors. The concentration of FT4 was stable during the observation period. The microspheres could be observed by histological methods on day 32. ¹³¹I-GMSs suppressed the growth of HepG2 in the nude mice and were retained in the tumor for a long period of time after injection. Direct intratumoral injection of ¹³¹I-GMSs offers a promising modality for the treatment of hepatocellular carcinoma.