Objective To investigate the effects of animal preparation on microPET imaging of tumor xenografts in nude mice and optimize the imaging protocol.Methods Thirty-six nude mice implanted with human epidermoid carcinoma A431cells were randomly divided into 6 groups. Group A: no fasting, room temperature (20℃ to 22℃), no anesthesia (leaving the animal awake for 60 min after the ¹⁸F-FDG injection), and ¹⁸F-FDG given by i.v. injection. Group B: Fasting (6 to 8 h), warming (30℃ to 32℃), anesthesia (inhaling 2% isoflurane anesthesia), and ¹⁸F-FDG given by i.v. injection. Group C: No fasting, warming, anesthesia, and ¹⁸F-FDG given by i.v. injection. Group D: Fasting, room temperature, anesthesia, and ¹⁸F-FDG given by i.v. injection. Group E: Fasting, warming, no anesthesia, and FDG given by i.v. injection. Group F: Fasting, warming, anesthesia, and ¹⁸F-FDG was given by i.p. injection. Serum glucose level was measured before FDG injection.%ID/g_max of the subcutaneous tumor, neck muscle, brown adipose tissue, brain, liver, kidney, myocardium, harderian gland of the groups A to F were measured after scanning. Results (1) The tumor ¹⁸F-FDG uptake was significantly inversely correlated with glycemia in the groups B, C and F (P<0.05). (2) The ¹⁸F-FDG uptakes in the brown adipose and muscle tissues in the group A were 8.03±1.29 and 16.07±5.20, respectively. The ¹⁸F-FDG uptakes in the brown adipose and muscle tissues in the group B were decreased by 71.98% and 81.84%, respectively, than that in the group A (P<0.05). The uptake in the cervical muscles was highest in the group A (16.07±5.20), and lowest in the group B, being 81.84% lower than that of the group A (P=0.000). The uptakes by brain, liver, kidney, myocardium and harderian gland were not significantly different among different groups. (3) The tumor-to-organ uptake ratio was lowest in the group A. The tumor-to-muscle, tumor-to-liver and tumor-to-brown fat uptake ratios were 6.5-fold, 1.29-fold and 4.76-fold increased, respectively, in the group B than that in the group A (P<0.05 for all). Under the experimental conditions of group B, the image contrast of tumor and organs was improved. (4) No significant differences were found for tumor ¹⁸F-FDG uptake by different routes of injection in the first scanning (P=0.364). After the second scanning, the ¹⁸F-FDG accumulation in the abdominal cavity by intraperitoneal injection led to a lower uptake of tumor and normal tissues. Significant differences were found for tumor ¹⁸F-FDG uptake by intraperitoneal injection between the first scanning and the second scanning (P=0.025). Conclusions Animal preparation has significant effects on the ¹⁸F-FDG biodistribution in normal tissues and the uptake in subcutaneously transplanted tumors. Fasting, warming, anesthesia, intravenous injection can improve the imaging quality and reproducibility.