Matrilins are adaptor proteins of the extracellular matrix involved in the formation of both collagen-dependent and collagen-independent filamentous networks. Although their molecular structure and binding partners have been characterized, the functional roles of the four matrilin family members in vivo are still largely unknown. Here, we show that matrilin 2, expressed in pre-myelinating Schwann cells during normal development, profoundly influences the behaviour of glial cells and neurons in vitro. When offered as a uniform substrate, matrilin 2 increased neurite outgrowth of dorsal root ganglia (DRG) neurons and enhanced the migration of both cell line- and embryonic DRG-derived Schwann cells. Vice versa, axonal outgrowth and cell migration were decreased in DRG cultures prepared from matrilin-2-deficient mice compared with wild-type (wt) cultures. In stripe assays, matrilin 2 alone was sufficient to guide axonal growth and, interestingly, axons favoured the combination of matrilin 2 and laminin over laminin alone. In vivo, matrilin 2 was strongly upregulated in injured peripheral nerves of adult wild-type mice and failure of protein upregulation in knockout mice resulted in delayed regrowth of regenerating axons and delayed time-course of functional recovery. Strikingly, the functional recovery 2 months after nerve injury was inferior in matrilin-2-deficient mice compared with wild-type littermates, although motoneuron survival, quality of axonal regeneration, estimated by analyses of axonal diameters and degrees of myelination, and Schwann cell proliferation were not influenced by the mutation. These results show that matrilin 2 is a permissive substrate for axonal growth and cell migration, and that it is required for successful nerve regeneration.