Radionuclide imaging of prostate-specific membrane antigen (PSMA) expression can be used for staging prostate cancer. The pseudo-peptide [99mTc]Tc-BQ0413 demonstrated high affinity and specificity to PSMA in preclinical evaluation. The purpose of this study was to clinically evaluate the safety and tolerability of a single administration of [99mTc]Tc-BQ0413 as well as study its biodistribution using SPECT to estimate dosimetry. [99mTc]Tc-BQ0413 was studied in a single-center diagnostic Phase I open-label exploratory study. Whole-body planar scintigraphy and SPECT/CT imaging were performed 2, 4, and 6 h after administration of 50, 100, or 150 μg (680 ± 140 MBq) of [99mTc]Tc-BQ0413 in five PCa patients per injected mass (NCT05839990). All injections of [99mTc]Tc-BQ0413 were well tolerated. The elimination of [99mTc]Tc-BQ0413 was predominantly renal. The stable physiological uptake of [99mTc]Tc-BQ0413 was observed in the lacrimal and salivary glands, liver, spleen, and kidneys for all tested peptide-injected masses. The average effective doses were 0.007 ± 0.001, 0.0049 ± 0.0003, and 0.0062 ± 0.0008 mSv/MBq for 50, 100, and 150 μg/injection, respectively. The radionuclide-associated dose burden per patient was 4-7 mSv/study for the given activity. Uptake of [99mTc]Tc-BQ0413 in primary tumors was identified in all patients and increased with the peptide-injected mass. Uptake in lymph nodes and bone metastases was the highest at 100 μg/injected mass. The highest tumor lesion/background ratios were observed 6 h after the administration of 100 μg of [99mTc]Tc-BQ0413. The results of the Phase I study showed that injections of [99mTc]Tc-BQ0413 were well tolerated, safe, and associated with low absorbed doses. Imaging using [99mTc]Tc-BQ0413 enabled the visualization of primary prostate cancer lesions as well as metastases in lymph nodes and bones.