Purpose: Previously we have shown that using sequential CT images is superior to sequential SPECT for nonrigid registration in three-dimensional (3D) targeted radionuclide therapy (TRT) dosimetry. However, sequential CTs are often not available due to radiation concerns. In this paper, we propose a virtual CT (vCT) method for attenuation and scatter correction, image registration, and segmentation for improved dosimetric accuracy with single CT acquisition. Methods: We used a population of nine XCAT phantoms with different In-111 Zevalin biokinetics and anatomical variations for the simulations. An analytical projector was used to simulate sequential SPECT/CT acquisitions for a medium energy general purpose collimator at 1, 12, 24, 72, and 144 h postinjection, modeling attenuation, scatter, and geometric collimator-detector response. The corresponding sequential attenuation maps of the phantoms served as real CT (rCT) images. For vCT generation, we investigated three registration methods, that is, (a) SPECT to SPECT; (b) SPECT to CT, and (c) CT to SPECT, and the optimal time point for single CT acquisition. Difference images and average normalized mean square errors (NMSE) were calculated between different vCTs and their corresponding rCTs. Absorbed dose and dose-volume histograms (DVHs) for critical organs were computed for the rCT, optimized vCT, and conventional single CT (1CT) protocols, respectively, for dosimetric analyses. Results: For vCT generation, SPECT to SPECT registration with a single CT acquired at the first time point shows the smallest difference and NMSE. For organ absorbed doses, the results of vCT were similar to those of rCT and were superior to 1CT, that is, −0.24 ± 1.56% vs −0.49 ± 1.76% vs −6.37 ± 5.63% for the liver, −1.05 ± 2.89% vs −0.69 ± 2.74% vs −4.87 ± 4.35% for kidneys, respectively. The results of DVHs also showed improvement for all organs using vCTs as compared to the conventional 1CT protocol. Conclusion: The optimized vCT method can effectively increase the TRT dosimetric results if there is only a single CT available in the sequential imaging protocol, reducing the substantial increase in radiation burden from repeated CT scans.