Mechanical signals influence axon growth of both CNS and PNS neurons. Type I collagen (Col-I) has been widely used as a substrate to study axon growth, since it emulates an in vivo environment. Here, we evaluated the influence of Col-I stiffness to the extent of sympathetic axon growth in three dimensions (3D). With this aim, superior cervical ganglion explants from neonatal rats were cultured in soft or stiff Col-I 3D matrices. Stiff Col-I 3D matrices were achieved following 4hs of treatment with glycolaldehyde (GA). Sympathetic axon growth was greatly influenced by Col-I stiffness both after 24 and 48hs. The main findings were: (1) explants seeded in soft Col-I matrices, formed radially symmetric halos composed of an intricate meshwork of neurites; (2) in stiff Col-I matrices, the neurites fasciculated, and formed smaller and apparently less dense halos; (3) neurites showed limited growth in stiff Col-I matrices evidenced by reduced length compared to those that grew in soft matrices and (4) a loss of growth cones in axons growing in Col-I stiff matrices was also evidenced. Here we showed that a mechanical property of the microenvironment, such as stiffness, can be less favorable to the growth of sympathetic axons. Together these results provide insights to the understanding of how growing neurons are affected when they interact mechanically with their environment, an issue that should be considered in tissue engineering and organoid research.