Objective:To investigate the possibility of quantitive analysis of 99mTc－HYNIC－PEG4－E[PEG4－c(RGDfk)]2 (99mTc－3P4－RGD2) uptake and tumor integrin αvβ3 expression in glioma bearing nude mice with microSPECT/CT imaging. Methods:microSPECT/CT imaging of 99mTc samples with different volume and radioactivity was carried out to assess the influence on radiocounting based upon volume and activity. 99mTc－3P4－RGD2 MicroSPECT/CT imaging was also carried out to glioma bearing nude mice. Tumor volume was determined in vivo by caliper,microCT,microSPECT and microSPECT/CT. The consistency between determined volume and actual volume was analyzed by Bland－Altman plots. Tumor uptake of 99mTc－3P4－RGD2 was quantified to estimate glioma expression level of integrin αvβ3 and confirmed by immunohistochemistry examination. Results:Linear relationship with R2 being 0.999 7 was found between the radioactivity counts from the SPECT/CT fusion images and those from γ－counter. Bias of microSPECT/CT measurements (%) compared with the reference ones was (-2.48 ± 8.17),less than that of microCT (15.04 ± 79.53,t = 5.60,P = 0.005),microSPECT (17.04 ± 36.27,t = 7.17,P = 0.002),respectively. MicroSPECT/CT can reflect vial and necrotic tumor volume and tumor uptake of 99mTc－3P－RGD2 was positively correlated with integrin αvβ3 expression with R2 being 0.95. Conclusion:MicroSPECT/CT imaging,which is more accurate in evaluating via tumor volume and integrin αvβ3 expression level,is of potential value for patients screening for integrin αvβ3 receptor targeted therapy and dose individualization.