The development of alternative therapies for melanoma treatment is of great interest as long-term tumour regression is not achieved with new targeted chemotherapies on selected patients. We previously demonstrated that radioiodinated heteroarylcarboxamide ([131I]ICF01012) induced a strong anti-tumoural effect by inhibiting both primary tumour growth and dissemination process in a B16BL6 melanoma model. In our study, we show that a single injection of [131I]ICF01012 (ranging from 14.8 to 22.2 MBq) was effective and associated with low and transient haematological toxicity. Concerning pigmented organs, cutaneous melanocytes and skin were undamaged. In 30% of treated animals, no histological alteration of retina was observed, and in the remaining 70%, damages were restricted to the optic nerve area. Using the Medical Internal Radiation Dose methodology, we determined that the absorbed dose in major organs is very low (<4 Gy) and that a delivery of 30 Gy to the tumour is sufficient for an effective anti-tumoural response. Molecular analyses of treated tumours showed a strong radiobiological effect with a decrease in proliferation, survival and pro-angiogenic-related markers and an increase in tumour suppressor gene expression, melanogenesis and anti-angiogenic markers. All these features are in accordance with a tumour cell death mechanism that mainly occurs by mitotic catastrophe and provide a better understanding of in vivo anti-tumoural effects of [131I] radionuclide. Our findings raise [131I]ICF01012 a good candidate for disseminated melanoma treatment and strongly support transfer of [131I]ICF01012 to clinical trial.