The standard of care for men with metastatic prostate cancer is androgen deprivation. Although initially all patients respond, resistance is inevitable and lethal castration-resistant prostate cancer ensues. Using patient-derived xenografts (PDX) of localised prostate tumours, we have identified a rare sub-population of ‘castration-tolerant’ prostate cancer cells that survive following castration. The aim of this study was to molecularly characterise human castrate-tolerant prostate cancer cells and identify novel targeting strategies to eliminate them and delay disease progression. To study the genomic features of castrate-tolerant cells, we enriched for prostate cancer cells from PDXs and subjected them to single cell isolation and RNA sequencing. In the absence of a definitive cell–surface antigen for prostate cancer, we have developed a panel of 16 fluorescent surface-markers to enrich for tumour cells from PDX grafts. Using the Fluidigm C1 platform, we captured and sequenced > 50 cells from pre- and post-castration prostate cancer PDXs. Sequencing of isolated single cells and pooled populations was performed using the Illumina HiSeq in rapid mode with 50 bp fragment sequencing chemistry (3Million reads/cell). Multidimensional scaling analysis showed that pre-castration and castrate-tolerant cells clustered separately, and that the response to castration is not uniform in all human cells, with variable degrees of heterogeneity seen within both groups. A unique gene set was identified in pre-castration versus castrate-tolerant luminal cells; we identified distinct changes in energy metabolism, including suppression of ATP production, that aid cell survival and detected a consistent upregulation of the retinoic acid signaling pathway, including upregulation of CRABP2 and RARRES3 expression in castrate-tolerant cells. This is the first study to report gene expression in single human prostate cells and revealed novel endocrine-related changes prior to and following androgen deprivation. Our data suggest that further and/or alterative hormone suppression may be effective in targeting castration-tolerant prostate cancer cells.