Prostate cancer preferentially metastasizes to the skeleton and abundant evidence exists that osteoblasts specifically support the metastatic process, including cancer stem cell niche formation. At early stages of bone metastasis, crosstalk of prostate cancer cells and osteoblasts through soluble molecules results in a decrease of cancer cell proliferation, accompanied by altered adhesive properties and increased expression of bone-specific genes, or osteomimicry. Osteoblasts synthesize a plethora of biologically active factors, which comprise the unique bone microenvironment. By means of quantitative real-time RT-PCR it was determined that exposure to the osteoblast secretome induced gene expression changes in prostate cancer cells, including the upregulation of osteomimetic genes such as BMP2, AP, COL1A1, OPG and RANKL. IL6 and TGFbeta1 signaling pathway components also became upregulated at early time points. Moreover, osteoblast-released IL6 and TGFbeta1 contributed to the upregulation of OPG mRNA in LNCaP. Thus, the earliest response of prostate cancer cells to osteoblast-released factors, which ultimately cause metastatic cells to assume an osteomimetic phenotype, involved activation of paracrine and autocrine IL6 and TGFbeta signaling. On the other hand, a microarray analysis showed that osteoblasts exposed to the secretome of prostate cancer cells exhibited gene expression alterations suggestive of repressed proliferation, decreased matrix synthesis and inhibited immune response, which together indicate enhanced preosteocytic differentiation. TGFbeta signaling, known to inhibit osteoblast maturation, was strongly suppressed, as shown by elevated expression of negative regulators, downregulation of pathway components and of numerous target genes. Transcriptional downregulation of osteoblast inhibitory molecules such as DKK1 and FST also occurred, with concomitant upregulation of the osteoinductive molecules ADM, STC1 and BMP2, and of the transcription factors CBFA1 and HES1, which promote osteoblast differentiation. Finally, the mRNA encoding NPPB, the precursor of a molecule implicated in the inhibition of TGFbetaeffects, in bone formation and in stem cell maintenance, became upregulated after coculture both in osteoblasts and in prostate cancer cells. These results provide an insight into potential mechanisms of dysregulated bone formation in metastatic prostate cancer, as well as mechanisms by which osteoblasts might enhance the invasive, osteomimetic and stem cell-like properties of the tumor cells. In particular, the differential modulation of TGFbetasignaling in prostate cancer cells and osteoblasts appears to merit further research.