Fibroblast growth factors (FGFs) are multifunctional signal molecules between cells, regulating the various physiological functions of the organism. FGF21 is a regulatory factor of the FGF family and has been postulated to play important roles in hair follicle development and hair follicle growth cycle. To evaluate the roles of FGF21, we had established a FGF21 knockout mouse model, using the CRISPR/Cas9 technology. We had constructed a FGF21 targeting vector and microinjected it with Cas9 mRNA and gRNA into fertilized ova of FVB mice. The gRNA was designed to target the exon 1 of the endogenous mouse FGF21 gene. Three lines of Fgf21 ^-/- mice were obtained from these experiments, and confirmed to harbor Fgf21 ^-/- genotypes and null expression phenotype, using DNA sequencing, qRT-PCR and Western blotting. FGF21 mRNA and FGF21 protein were not detected in tissues of these Fgf21 ^-/- mice. Depilation and histochemistry analyses showed that the Fgf21 ^-/- mice had lower body weight, slower hair regrowth and poorer hair quantities and smaller hair follicles diameters, as compared to WT mice. The Fgf21 ^-/- mice reported here could provide a useful genetic model for future studies of FGF21 functions in hair follicle development and hair follicle growth cycle.