The objective of this study was to investigate the therapeutic potential of ¹³¹I added to doxorubicin therapy in multidrug resistance (MDR) mouse colon cancer coexpressing the MDR1 small hairpin RNA (shRNA) and human sodium iodide symporter (hNIS) gene in a single gene construct and to visualize the antitumor effects using molecular nuclear imaging. HCT-15 coexpressing shRNA for MDR1 gene (MDR1 shRNA) and hNIS gene with a single construct was established (referred to as MN61 cell). Inhibition of P-gp function by MDR1 shRNA and functional activity of hNIS gene was assessed using a ⁹⁹(m)Tc sestamibi uptake and ¹²⁵I uptake, respectively. Cytotoxic effects by a combination of doxorubicin and ¹³¹I were determined in parental (HCT-15) or MN61 cells using an in vitro clonogenic assay. Therapeutic effect of either combination therapy (doxorubicin and ¹³¹I) or single therapy (doxorubicin or ¹³¹I alone) was evaluated by tumor volume measurement. ⁹⁹(m)Tc-sestamibi, ¹²³I, and ⁹⁹(m)Tc-pertechnetate images of mice were acquired to evaluate functional assessment in vivo. Cellular uptake of ⁹⁹(m)Tc-sestamibi and ¹²⁵I was approximately 2-fold and 100-fold higher in MN61 cells than in parental cells, respectively. Combination of ¹³¹I and doxorubicin resulted in higher cytotoxcity in MN61 cells as compared with parental cells. Scintigraphic imaging showed higher uptake of ⁹⁹(m)Tc-sestamibi and ¹²³I in MN61 tumor as compared with parental tumor. In mice treated with doxorubicin, there was a slight delay in tumor growth in the MN61 tumor but not in the parental tumor. Cancer treatment with ¹³¹I or doxorubicin induced a rapid reduction of tumor volume in the MN61 tumor but not in the parental tumor. Combination therapy further generated a rapid reduction of tumor volume as compared with ¹³¹I therapy alone (p < 0.05). A combination hNIS mediated radioiodine gene therapy added to MDR1 shRNA treatment improved the effects of cancer treatment in a MDR cancer model and could enable visualization of the antitumor effects with nuclear imaging.