Prostate cancer cells reside in a complex stromal microenvironment often referred to as “reactive” stroma which is a critical component of prostate cancer initiation and progression. The mounting evidence for its critical nature has led to increased interest in this niche as a target for new therapeutic approaches. Cancer associated fibroblasts (CAFs) play a key role in this niche regulating the tumour microenvironment. Factors secreted by prostate cancer cells can ‘activate’ non-malignant associated fibroblasts to become CAFs. KLK4 is over-expressed in both localised and bone metastatic prostate cancer and so has the capacity to act as a paracrine factor on the surrounding stroma. The Aim of this study was to elucidate the role of KLK4 in tumour-stroma cross-talk by identifying its substrates in the prostate cancer lines, LNCaP and PC3 (also derived from a bone metastasis) and in the prostate (stromal) fibroblast line WPMY-1. This was accomplished at the protein level utilising the ‘PROtein TOpography Migration Analysis Platform’ (Dix et al., Cell, 2008). Gene expression changes following KLK4 treatment were also assessed by gene microarray analysis. We identified 50 putative novel KLK4 protein substrates, 11 of which directly interact with the growth factor TGFβ1. Strikingly, the most enriched pathway (based on DAVID analysis, p<0.01) following transcriptome analysis of the KLK4-treated cells was the TGFβ1 pathway. KLK4-treated fibroblasts also expressed elevated levels of a number of genes consistent with a CAF genotype. These findings suggest that KLK4 is a critical regulator of the reactive stromal niche, via the TGFβ1 pathway, and a potential novel therapeutic target for prostate cancer.