EphrinB2 (Efnb2) is a membrane-bound ligand for the receptor tyrosine kinase EphB4. Their cell contact-dependent interaction activates signalling through both receptor (forward) and ligand (reverse). We previously reported that in vivo deletion of Efnb2 in early osteoblasts delays bone mineralisation, and in late osteoblasts and osteocytes increases mineral content and bone fragility. The latter observation was associated with accumulation of autophagic vesicles in osteocytes and fewer intracellular acidic vesicles (lysosomes). Here, we sought to determine how EphrinB2:EphB4 signalling controls lysosome trafficking and mineralisation. To determine whether EphrinB2-deficient cells can form lysosomes, control and EphrinB2-deficient (ΔEfnb2) osteoblast-like Ocy454 cells were treated in vitro with the mTORC1 inhibitor INK128. Lysosome content was assessed by live-cell fluorescence imaging.  Both genotypes exhibited a 2-fold increase in lysosomes. When exocytosis was blocked with vacuolin-1, lysosomes accumulated in both control and ΔEfnb2 cells, but at 30% higher levels in ΔEfnb2 cells. This suggests EphrinB2-deficient osteoblasts form more lysosomes, but release them rapidly, leading to low intracellular lysosome numbers. Stimulation of either EphB4 or EphrinB2 signalling in wildtype Ocy454 cells by exogenous ligand or receptor reduced lysosomes by 15-20%, and completely blocked INK128-induced lysosome formation, confirming that EphrinB2:EphB4 signalling inhibits lysosome formation. Increased release in ΔEfnb2 cells was confirmed by transmission electron microscopy: dense vesicles were observed in the cytoplasm of control cells but not in ΔEfnb2 cells, where they instead were budding from the cell membrane. To test whether vesicles released by ΔEfnb2 cells stimulate mineralisation, control or ΔEfnb2 cells were supplied in Transwells to mineralising wildtype Ocy454 cells. While addition of control cells reduced mineralisation, addition of ΔEfnb2 cells increased mineralisation. Finally, proteomics analysis of extracellular vesicles from ΔEfnb2 cells indicated that, in response to mineralising conditions they secreting vesicles containing more collagen 1 isoforms (a1/a2) than controls, along with higher levels of collagen processing and crosslinking enzymes (e.g. osteomodulin, periostin and lysyl oxidase homologs 2/3). In summary, EphrinB2:EphB4 signalling limits lysosome formation in osteoblasts. In EphrinB2 deficiency, increased lysosome production and trafficking lead to greater release of collagen and stimulate mineral accrual. This suggests a new control mechanism by which osteoblasts limit bone mineralisation.