The main aim of this study was to investigate the possible association between 18F-choline uptake and histopathological features of prostate biopsies such as the Gleason Group and the expression of both epithelial to mesenchymal transition (vimentin) and bone mineralization (bone morphogenetics protein (BMP)-2, runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor-κB ligand (RANKL), vitamin D receptor (VDR), and pentraxin 3 (PTX3) in situ biomarkers. To this end, we enrolled 79 consecutive prostate cancer patients that underwent both the 18F-choline PET/CT analysis and the prostate bioptic procedure. The standardized uptake value (SUV) average values were collected from 18F-choline PET/CT analysis whereas Gleason Group and immunostaining data were collected from paraffin-embedded sections. Histological classification showed a heterogenous population including both low/intermediate and high-grade prostate cancers. A significant increase of 18F-choline uptake in high-grade prostate lesions (Gleason Score ≥8) was found. Also, linear regression analysis showed a significant correlation between 18F-choline uptake and the number of vimentin, RANKL, VDR, or PTX3 positive prostate cancer cells. Conversely, we observed no significant association between 18F-choline uptake and the expression of bone biomarkers involved in the early phases of osteoblast differentiation (BMP-2, RUNX2). In conclusion, results here reported can lay the foundation for the use of 18F-choline positron emission tomography (PET)/computed tomography (CT) as a diagnostic tool capable of identifying high-grade prostate cancer lesions expressing bone biomarkers.