Acrylic acid/fibrin hydrogel can mechanically stimulate cells when an external electrical field is applied, enabling them to migrate and align throughout the depth of the gel. The ability of electro-responsive polyacrylic acid (PAA)/fibrin hydrogel to promote collagen production and remodeling has been investigated by three-dimensional (3D) culturing and conditioning of smooth muscle cells (SMCs). SMCs-seeded hydrogels were subjected to an alternating electrical field (0.06 V/mm) for 2 h for one, two, or three times per week during 4 weeks of culturing. Fluorescent images of collagen structure and accumulation, assessed by CNA-35 probe, showed increased collagen content (>100-fold at 1× stimulation/week) in the center of the hydrogels after 4 weeks of culture. The increase in collagen production correlated with increasing extracellular matrix gene expression and resulted in significantly improved mechanical properties of the stimulated hydrogels. Matrix metalloproteinase (MMP)-2 activity was also significantly enhanced by stimulation, which probably has a role in the reorganization of the collagen. Short stimulation (2 h) induced a favorable response in the cells and enhanced tissue formation and integrity of the scaffold by inducing collagen production. The presented set up could be used for conditioning and improving the functionality of current tissue-engineered vascular grafts.