Marshall-Smith syndrome (MSS) is a congenital disorder characterised by developmental delay, short stature, respiratory difficulties, distinctive facial features, skeletal abnormalities (such as kyphoscoliosis, dysostosis and osteopenia) and delayed neural development, and is due to heterozygous mutations that are clustered in exons 610 of the transcription factor nuclear factor I/X (NFIX) gene. These frameshift and splice-site NFIX variants result in the production of aberrant transcripts that escape nonsense mediated mRNA decay and lead to the production of dominant negative mutant NFIX protein. To elucidate the in vivo effects of mutant NFIX, CRISPR-Cas9 was used to generate a mutant mouse model with a frameshift deletion of 2 nucleotides (Nfix Del2) in Nfix exon 7. All animal studies were ethically performed under an approved UK Home Office Animal License. Nfix+/Del2 mice were viable, normal and fertile but NfixDel2/Del2 mice had significantly reduced viability (P<0.002), and died at 23 weeks of age. Phenotypic characterisation of the 23 weeks NfixDel2/Del2 mice showed that, compared to Nfix+/+ and Nfix+/Del2 mice, NfixDel2/Del2 mice had significantly reduced: growth rate (0.8-fold, P<0.05); tail length (0.9-fold, P<0.001); lean (0.8-fold, P<0.0001) and fat (0.4-fold, P<0.0001) mass; weight (0.8-fold, P<0.0001); and total tissue mass (0.8-fold, P<0.0001). Moreover, >30% (P<0.0001) of NfixDel2/Del2 mice had kyphosis compared to <10% Nfix+/+ and Nfix+/Del2 mice, and micro-CT scans of the lumbar and thoracic vertebrae revealed NfixDel2/Del2 mice to have osteopenia. In addition, plasma biochemistry analysis of NfixDel2/Del2 mice revealed that, compared to Nfix+/+ and Nfix+/Del2 mice, NfixDel2/Del2 mice had significantly: increased plasma urea (1.4-fold, P<0.0001) and total bilirubin (1.5-fold, P<0.0001) concentrations; and alkaline phosphatase activity (1.5-fold, P<0.0001); but decreased procollagen type 1 N-terminal propeptide (0.8-fold, P<0.01) concentrations. Thus, NfixDel2/Del2 mice provide a useful model for studying the skeletal, renal and hepatic effects of mutant NFIX and the mechanisms underlying the aetiology of MSS.