Neurovirulence is one of the pathological complications associated with vaccinia virus (VV) infection/vaccination. Although the viral N1L protein has been identified as the neurovirulence factor, none of the host N1L-interacting factors have been identified so far. In the present study, we identified N1L-interacting proteins by screening a human brain cDNA expression library with N1L as a bait protein in a yeast two-hybrid analysis. The analysis revealed that N1L interacts with human brain-originated cellular basement membrane-associated chondroitin sulfate proteoglycan (bamacan). The N1L-binding domain of bamacan was mapped to its C-terminal 227 amino acids. The N1L-bamacan interaction was further confirmed in both VV-infected and N1L-transfected mammalian cells. Following the confirmation of the protein interactions by coimmunoprecipitation experiments, confocal microscopic analysis revealed that N1L colocalizes with bamacan both in VV-infected B-SC-1 cells as well as in mice neuronal tissue. Furthermore, a human neural cell line, which expresses bamacan to moderately elevated levels relative to a non-neural cell line, supported enhanced viral growth. Overall, these studies clearly suggest that bamacan interacts with the VV-N1L and such interactions seem to play a positive role in promoting the viral growth and perhaps contribute to the virulence of VV in neural cells.