^99mTc-3PRGD₂ is a new SPECT radiotracer for several tumor imaging with high uptake where integrin α_vβ₃ is highly expressed. This pilot study was to assess the safety, biodistribution and radiation dosimetry of ^99mTc-3PRGD₂ in healthy volunteers. The 10 healthy male volunteers were injected with ^99mTc-3PRGD₂ (786.7±55.8 MBq, 19.1–24.2 mCi). Baseline measurements of vital signs, laboratory safety tests and 12-lead electrocardiogram were recorded before and after injection. Blood and urine samples were collected and radiation counts were obtained at various time points. Whole-body scans and ROIs of identified organs were used for visual analysis and estimating the radiation dosimetry. No adverse reactions were found during the study. ^99mTc-3PRGD₂ exhibited a rapid clearance from the blood with less than 45% of the initial dosage at 10 min after injection and gradual increasing radioactivity in urine with (52.9±6)% of original dose at 1440 min. The whole-body imaging showed high radioactive accumulation in bladder. And the highest ^99mTc-3PRGD₂ uptake was found in the kidneys (3.50×10⁻² mSv/MBq). The ^99mTc-3PRGD₂ exhibited good pharmacokinetic properties and little radiation burden. This study showed that ^99mTc-3PRGD₂ would be a safe and attractive SPECT agent in clinic applications.