Angiogenesis is essential to tissue reconstitution, is sensitive to mechanical stresses, and currently represents one of the major challenges in tissue engineering. The pro-angiogenic matrix metalloprotease-2 (MMP-2) is upregulated in mechanically loaded mesenchymal stem cells (MSCs). Therefore, MMP-2 may provide a regulating link between angiogenesis and the surrounding mechanical conditions. This study aimed to modulate MMP-2 levels by mechanical loading of MSCs embedded in a three-dimensional matrix as well as to investigate the mechanism of MMP-2 regulation along with its contribution to angiogenesis stimulation. MMP-2-inducing conditions (30% compression, 1 Hz, 72 h) were defined after varying loading parameters. Addition of the Golgi-disturbing agent Brefeldin A suppressed this mechanical upregulation of MMP-2. Analysis of enzymatic activities demonstrated an enhancement of pro-MMP-2, mature MMP-2, and tissue inhibitor of metalloproteases-2. Further, mechano-regulation of MMP-14 and mature MMP-2 was dependent upon the activity of furin, a proprotein processing endoprotease. Angiogenesis was stimulated by conditioned media from MSCs loaded at inducing conditions. This augmentation of angiogenesis was hindered by inhibition of pro-MMP-2 and mature MMP-2. In conclusion, mechanical stimulation of MSCs in a three-dimensional matrix induces pro-MMP-2 secretion and MMP-2 activation, potentially via the activation complex consisting of MMP-2/-14/tissue inhibitor of metalloproteases-2. Mechano-regulated pro-MMP-2 and mature MMP-2 seem to contribute to angiogenesis stimulation. Thus, an application of these loading parameters could augment vascularization of tissue-engineered constructs based on the described MMP-2-dependent mechanism.