Objective To construct v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) knockout mice by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), and explore the impact brought by this gene deletion on the urethral development. Methods The F0 Mafb knockout C57BL/6J mice were constructed according to the principle of CRISPR/Cas9 technology. F0 mice identified by polymerase chain reaction and gene sequencing were mated with wild-type C57BL/6J mice to obtain F1 Mafb knockout heterozygous (Mafb^+/-) mice. Mafb knockout homozygous (Mafb^-/-) fetal mice were obtained by mating between Mafb^+/- mice. The penile tissues of male Mafb^-/- fetal mice were collected. The expression of Mafb protein in the penile tissues and the expression of epithelial cadherin (E-cadherin) and α-smooth muscle actin (α-SMA) in the urethral seam were detected by immunofluorescence. The histological morphology of the penile tissues was observed by scanning electron microscopy and paraffin section hematoxylin-eosin (H-E) staining. Results The Mafb^+/- mice were successfully constructed and bred, and male Mafb^-/- fetal mice were obtained. Immunofluorescence showed scarce expression of Mafb protein in the penises of the male Mafb^-/- fetal mice; compared with wide type mice, the expression of E-cadherin was downregulated and α-SMA was upregulated in the urethral seam. Scanning electron microscopy and H-E staining showed that the phenotype of male Mafb^-/- fetal mice was hypospadias. Conclusion The Mafb knockout mouse model is successfully constructed. The knockout of Mafb can lead to the hypospadias phenotype and change the expression levels of E-cadherin and α-SMA on the urethral seam. This model lays a foundation for further study on the role of Mafb in the pathogenesis of hypospadias.