Cancer cells undergo metabolic reprogramming to meet increased bioenergetic demands. Studies in cells and mice have highlighted the importance of oxidative metabolism and lipogenesis in prostate cancer, however, the broader metabolic landscape of human prostate cancer remains unclear. The aim of this study was to study metabolic substrate utilisation in patient-derived xenografts (PDXs) representing different stages of prostate cancer disease progression. The methods included radiometric (14C) and stable (13C) isotope tracing assays in precision-cut PDX slices derived from 35 individual tumours, including nine benign, 13 localised, and 13 metastatic prostate tissues. Glucose, glutamine, and fatty acid oxidation was variably up-regulated in cancerous PDXs compared to benign PDXs, while lactate oxidation was unchanged. De novo lipogenesis (DNL) and storage of free fatty acids into phospholipids and triacylglycerols were increased in malignant PDXs. There was no difference in substrate utilisation between localised and metastatic PDXs and hierarchical clustering revealed marked metabolic heterogeneity across all PDXs. Mechanistically, glucose utilisation was mediated by acetyl-CoA production rather than carboxylation of pyruvate, while glutamine entered the TCA cycle through transaminase reactions before being utilised via oxidative or reductive pathways. Blocking fatty acid uptake or fatty acid oxidation with pharmacological inhibitors was sufficient to reduce cell viability in all PDX-derived organoids (PDXOs) examined, whereas blockade of DNL, or glucose or glutamine oxidation induced variable and limited therapeutic efficacy in PDXOs. These findings demonstrate that human prostate cancer, irrespective of disease stage, can effectively utilise all metabolic substrates, albeit with marked heterogeneity across tumours. We also confirm that fatty acid uptake and oxidation are targetable metabolic vulnerabilities in human prostate cancer.