Objective To prepare 99 Tcm-HYNIC-c( isoDGRKy) as a SPECT/CT imaging molecu-lar probe targeting integrin αvβ3 , and evaluate its biodistribution and feasibility on SPECT/CT imaging for integrinαvβ3-positive tumor in U87MG human glioma xenograft mouse models. Methods The bifunctional chelator HYNIC was conjugated to c( isoDGRKy) , and tricine and TPPTS were used as coligands for 99 Tcm labeling to prepare 99 Tcm-HYNIC-c( isoDGRKy) . The radiochemical purity and stability of the product were measured. The expression of integrin αvβ3 and binding affinity ( half maximal inhibitory concentration, IC50 ) of c ( isoDGRKy ) was detected in U87MG cells by cell experiments in vitro. Biodistribution and SPECT/CT imaging of 99 Tcm-HYNIC-c( isoDGRKy) including blocking experiments were performed respec-tively in nude mice bearing U87MG human glioma xenografts. Results The radiochemical purity of 99 Tcm-HYNIC-c( isoDGRKy) was over 99%, and was still over 99% after 4 h incubation in saline at room temper-ature. Flow cytometry assay showed that U87MG cells were integrinαvβ3-positive ( expressive rate:70%) . The IC50 of c(isoDGRKy) was 6.67×10-8 mol/L. Biodistribution results showed 99Tcm-HYNIC-c(isoDGRKy) with a rapid clearance from blood was excreted mainly via the kidneys. The 99 Tcm-HYNIC-c( isoDGRKy) uptake values in U87MG tumors were (7.31±1.42) and (1.09±0.11) %ID/g at 15 and 45 min post-injection re-spectively, and tumor-to-muscle ratio reached 5.01±1.47 at 15 min post-injection. The tumors were clearlyvisualized with low background from 0.5 to 1 h post-injection in tumor bearing mice. In the blocking experi-ment, the tumor was barely visualized after co-injection of excess cold c(RGDfK) peptide with 99Tcm-HYNIC-c(isoDGRKy). Conclusions 99Tcm-HYNIC-c(isoDGRKy) may be easily and steadily prepared. It may be a RGD-like promising SPECT/CT imaging probe for integrinαvβ3-positive tumor.