The fundamental role of epigenetic regulatory mechanisms involved in neuroplasticity and adaptive responses to traumatic brain injury (TBI) is gaining increased recognition. TBI-induced neurodegeneration is associated with several changes in the expression and activity of various epigenetic-regulatory enzymes, including histone deacetylases (HDACs). In this study, PET/CT with 6-((¹⁸F)trifluoroacetamido)-1-hexanoicanilide ([¹⁸F]TFAHA) to image the spatial and temporal dynamics of HDACs class IIa enzymatic activity in the brain of rats subjected to a weight drop model of diffuse, non-penetrating brain injury (termed mild TBI;​ mTBI), that was validated by histopathological and immunohistochemical analyses of brain tissue sections for the localization and magnitude of expression of heat shock protein-70 kDa (HSP70), amyloid precursor protein (APP), cannabinoid receptor-2 (CB2), and ionized calcium binding adapter protein-1 (IBA1), and histone deacetylases 4 and 5 (HDAC4 and HDAC5). As compared to baseline, the expression-activities of HDAC4 and of HDAC5 were downregulated in the hippocampus, nucleus accumbens, paraventricular thalamic nuclei, and substantia nigra at 1-3 days post mTBI, and remained low at 7-8 days post mTBI. Reduced levels of HDAC4 and HDAC5 expression observed in neurons of these brain structures post mTBI were associated with the reduced nuclear and neuropil levels of HDAC4 and HDAC5 with predominant shift to perinuclear localization of these enzymes. These results support the rationale for the development of therapeutic strategies aimed to upregulate the enzymatic activity of HDACs class IIa post-TBI. PET/CT(MRI) with [¹⁸F]TFAHA can facilitate the development and clinical translation of novel therapeutic approaches to upregulate the expression and activity of HDACs class IIa enzymes in the brain after TBI.