The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood. We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. First, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate–oxaloacetate–citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate–malate conversion. Our results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa. •Targeted metabolomics and stable isotope tracer analysis were optimized in mouse and human prostate organoids.•Organoids recapitulate the unique citrate-secretory phenotype of the prostate.•Glutamine fuels citrate synthesis for secretion by glutaminolysis and reductive carboxylation.•Aspartate enters the TCA cycle at different entry points in mouse and human prostate organoids for citrate production.•We revealed a much more complex TCA cycle in the prostate than originally anticipated.