Purpose <p>Colorectal cancer (CRC) is the second most fatal cancer in the world, accounting for 9.4% of all cancer-related deaths. Radiotherapy is a common treatment for late stages of cancer. However, the radiation therapy could cause unwanted damage to the surrounding healthy tissues. Hence, this study aims to develop holmium-166 (¹⁶⁶Ho)-loaded calcium carbonate (CC) nanoparticles as therapeutic agent for selective internal radiation therapy of CRC.</p> Methods <p>The CC nanoparticles were synthesized using co-precipitation. The CC nanoparticles was then loaded with ¹⁶⁵Ho through ionic exchange reactions between Ca²⁺ and ¹⁶⁵Ho³⁺ ions. The [¹⁶⁵Ho]-loaded CC nanoparticles were then neutronactivated in a nuclear reactor, converting ¹⁶⁵Ho to ¹⁶⁶Ho. Physicochemical characterization of the developed nanoparticles before and after neutron activation was conducted using FESEM, EDX spectroscopy and zetasizer. The presence of radionuclide impurities and in vitro radionuclide retention efficiency of the neutron activated nanoparticles were also determined. The toxicity of the nanoparticles on HT-29 cells was evaluated using MTT assay. The imaging potential of the [¹⁶⁶Ho]-loaded CC nanoparticles were investigated using static gamma camera.</p> Results <p>The [¹⁶⁵Ho]-loaded CC nanoparticles were having the size of 173.03 ± 19.06 nm with a zeta potential of -21.6 ± 0.95 mV. The [¹⁶⁶Ho]-loaded CC nanoparticles achieved a specific activity 1.85 ± 0.56 GBq/g after 5&#xa0;h of neutron activation. Gamma spectroscopy confirmed no radionuclide impurities observed in the nanoparticles. The gamma camera was able to detect the [¹⁶⁶Ho]-loaded CC nanoparticles. No significant toxicity of the [¹⁶⁵Ho]-loaded CC nanoparticles was observed on HT-29 cells. The nanoparticles show an excellent radionuclide retention efficiency of more than 99% in PBS solution of 7.4 and 5.5 over a duration of 216&#xa0;h.</p> Conclusions <p>The [¹⁶⁶Ho]-loaded CC nanoparticles were developed in this study as a potential internal radiation therapeutic agent for CRC. Further studies will be conducted to evaluate therapeutic efficacy on the CRC cell lines.</p>