We aim to characterize the metabolic changes associated with early response to radiation therapy in a prostate cancer mouse model by 2-deoxy-2-[F]fluoro-d-glucose ([F]FDG) and [C]acetate ([C]ACT) positron emission tomography, with nuclear magnetic resonance (NMR) metabolomics corroboration. [F]FDG and [C]ACT PET were performed before and following irradiation (RT, 15Gy) for transgenic adenocarcinoma of mouse prostate xenografts. The underlying metabolomics alterations of tumor tissues were analyzed by using ex vivo NMR. The [F]FDG total lesion glucose (TLG) of the tumor significant increased in the RT group at Days 1 and 3 post-irradiation, compared with the non-RT group (p < 0.05). The [C]ACT maximum standard uptake value (SUVmax) in RT (0.83 ± 0.02) and non-RT groups (0.85 ± 0.07) were not significantly different (p > 0.05). The ex vivo NMR analysis showed a 1.70-fold increase in glucose and a 1.2-fold increase in acetate in the RT group at Day 3 post-irradiation (p < 0.05). Concordantly, the expressions of cytoplasmic acetyl-CoA synthetase in the irradiated tumors was overexpressed at Day 3 post-irradiation (p < 0.05). Therefore, TLG of [F]FDG in vivo PET images can map early treatment response following irradiation and be a promising prognostic indicator in a longitudinal preclinical study. The underlying metabolic alterations was not reflected by the [C]ACT PET.