Vascular endothelial growth factor (VEGF) is a neurotrophic factor essential for maintenance of motor neurons. Loss of this factor produces a phenotype similar to amyotrophic lateral sclerosis (ALS). We recently showed that ALS-producing mutations of Cu/Zn-superoxide dismutase (SOD1) disrupt post-transcriptional regulation of VEGF mRNA, leading to significant loss of expression [Lu et al., J. Neurosci.27 (2007), 7929]. Mutant SOD1 was present in the ribonucleoprotein complex associated with adenine/uridine-rich elements (ARE) of the VEGF 3′-untranslated region (UTR). Here, we show by electrophoretic mobility shift assay that mutant SOD1 bound directly to the VEGF 3′-UTR with a predilection for AREs similar to the RNA stabilizer HuR. SOD1 mutants A4V and G37R showed higher affinity for the ARE than L38V or G93A. Wild-type SOD1 bound very weakly with an apparent Kd 11- to 72-fold higher than mutant forms. Mutant SOD1 showed an additional lower shift with VEGF ARE that was accentuated in the metal-free state. A similar pattern of binding was observed with AREs of tumor necrosis factor-α and interleukin-8, except only a single shift predominated. Using an ELISA-based assay, we demonstrated that mutant SOD1 competes with HuR and neuronal HuC for VEGF 3′-UTR binding. To define potential RNA-binding domains, we truncated G37R, G93A and wild-type SOD1 and found that peptides from the N-terminal portion of the protein that included amino acids 32-49 could recapitulate the binding pattern of full-length protein. Thus, the strong RNA-binding affinity conferred by ALS-associated mutations of SOD1 may contribute to the post-transcriptional dysregulation of VEGF mRNA.