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Aims: A non-coding GGGGCC (G4C2) repeat expansion in C9orf72 gene is the most frequent cause of Amyotrophic Lateral Sclerosis (ALS). RNAs containing the repeat, which accumulate as RNA foci in the nucleus and/or cytoplasm of affected cells, bind to various RNA-binding proteins, including several translational regulators, possibly impairing their function. Thus, in order to understand the mechanisms of C9orf72 toxicity, we investigated the functional consequences of G4C2 expression on mRNA trafficking and protein translation. Methods: HeLa cells transfected with (G4C2)31 repeats were used as cellular model. Fluorescence in situ hybridization (FISH), coupled to immunofluorescence analysis, was used to assess RNA foci formation, mRNA distribution and localization of repeat-binding partners. Global protein synthesis was monitored with the SUnSET method. Results: Expression of (G4C2)31 repeats, which induces the formation of nuclear RNA foci, causes stress granules formation, reduction of protein translation and a marked nuclear accumulation of poly-adenylated mRNAs, suggesting that defects in nuclear mRNA export might affect its trafficking and translation. Indeed, C9orf72 repeats interacts with the mRNA export factor NXF1, suggesting a direct interference with NXF1 function. Moreover, overexpression of a dominant negative form of NXF1 reproduces key phenotypes characterizing cells expressing the repeat. Interestingly, modulation of NXF1 expression alters G4C2 RNA foci formation, suggesting a crucial role of this factor in regulating both total mRNA and C9orf72 RNA cellular trafficking. Conclusions: G4C2 expression impairs the NXF1-mediated mRNA export pathway, and the resulting nuclear mRNA retention might affect translation efficiency and contribute to the pathogenesis of C9orf72-ALS.