A dynamic interplay between prostate cancer (PCa) cells and reactive bone stroma modulates the growth of metastases within the bone microenvironment. Of the stromal cells, metastasis-associated fibroblasts (MAFs) are known to contribute but are the least studied cell type in PCa tumour progression. It is the aim of the current study to establish a biologically relevant 3D in vitro model that mimics the cellular and molecular profiles of MAFs found in vivo. Using 3D in vitro cell culture models, the bone-derived fibroblast cell line, HS-5, was treated with conditioned media from metastatic-derived PCa cell lines, PC3 and MDA-PCa 2b, or mouse-derived fibroblasts 3T3. Two corresponding reactive cell lines were propagated: HS5-PC3 and HS5-MDA, and evaluated for alterations in morphology, phenotype, cellular behaviour, plus protein and genomic profiles. HS5-PC3 and HS5-MDA displayed distinct alterations in expression levels of N-Cadherin, non-functional E-Cadherin, alpha-smooth muscle actin (α-SMA), Tenascin C, and vimentin, along with transforming growth factor receptor expression (TGF β R1 and R2), consistent with subpopulations of MAFs reported in vivo. Transcriptomic analysis revealed a reversion of HS5-PC3 towards a metastatic phenotype with an upregulation in pathways known to regulate cancer invasion, proliferation, and angiogenesis. The exploitation of these engineered 3D models could help further unravel the novel biology regulating metastatic growth and the role fibroblasts play in the colonisation process.