Smooth muscle tissue is characterized by aligned structures, which is critical for its contractile functions. Smooth muscle injury is common and can be caused by various diseases and degenerative processes, and there remains a strong need to develop effective therapies for smooth muscle tissue regeneration with restored structures. To guide cell alignment, previously cells were cultured on 2D nano/microgrooved substrates, but such method is limited to fabricating 2D aligned cell sheets only. Alternatively, aligned electrospun nanofiber has been employed as 3D scaffold for cell alignment, but cells can only be seeded post fabrication, and nanoporosity of electrospun fiber meshes often leads to poor cell distribution. To overcome these limitations, we report aligned gelatin-based microribbons (mu RBs) as macroporous hydrogels for guiding smooth muscle alignment in 3D. We developed aligned mu RB-like hydrogels using wet spinning, which allows easy fabrication of tissue-scale (cm) macroporous matrices with alignment cues and supports direct cell encapsulation. The macroporosity within mu RB-based hydrogels facilitated cell proliferation, new matrix deposition, and nutrient diffusion. In aligned mu RB scaffold, smooth muscle cells showed high viability, rapid adhesion, and alignment following mu RB direction. Aligned mu RB scaffolds supported retention of smooth muscle contractile phenotype, and accelerated uniaxial deposition of new matrix (collagen I/IV) along the mu RB. In contrast, cells encapsulated in conventional gelatin hydrogels remained round with matrix deposition limited to pericellular regions only. We envision such aligned mu RB scaffold can be broadly applicable in growing other anisotropic tissues including tendon, nerves and blood vessel. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1064-1071, 2016.