Tumour progression relies on interactions between transformed tumour cells and untransformed cells in the tumour microenvironment (TME), co-opted by tumours to promote blood supply, tumour growth and immune evasion. Eph receptors and their cell-bound ephrin ligands control cell-cell interactions guiding vascular and neural patterning during normal development, and re-emerge in tumours and the TME. EphA3 is over-expressed in a range of solid tumour types, and we previously found this expression was not only in tumour cells, but more commonly in supportive stromal and vascular tissues in human tumour samples and in mouse xenografts. To investigate whether EphA3 expression in the TME supports tumour growth, we generated mice with transgenic inducible shRNA expression to confer knockdown of EphA3 in the TME. Mesenchymal stromal progenitor cells (MSCs) from mice with shRNA-induced EphA3 knockdown exhibited reduced angiogenic capacity, and EphA3 knockdown in mice bearing syngeneic tumour allografts was associated with a reduction of EphA3+ stromal cells in tumours, and a decrease in vessel formation and tumour growth. EphA3 was particularly expressed on perivascular myofibroblast-like cells, both in mouse tumours and in human breast and pancreatic cancers, as evident from single cell RNA sequencing analysis. Targeting these cells with a novel anti-EphA3 antibody-drug conjugate inhibited tumour growth in mice, demonstrating potential therapeutic utility. Our results thus indicate that stromal EphA3 expression plays an important and targetable role in supporting tumour angiogenesis and tumour growth.