DnaJ homolog, subfamily B, member 4 (DNAJB4) is a member of the heat shock protein 40 chaperone family, and it is highly expressed in striated muscles. Although DNAJB6 is known to cause LGMDD1, no human disease related to DNAJB4 has been reported so far. Here, we report one family including six affected individuals. The initial symptom was hand grip weakness from around 30 years of age, then they developed lower limb weakness from their 40’s. Muscle imaging showed a characteristic pattern of involvement with rectus femoris and hamstrings affected. Muscle pathology showed amorphous cytoplasmic inclusions and rimmed vacuoles. Using whole exome sequencing, we identified a heterozygous missense variant, c.270T>A (p.F90L) in all affected members examined showing co-segregation with the disease within the family. This variant was not listed in any public databases and the substituted amino acid position is highly evolutionarily conserved. We generated knock-in mice for the F90L mutation. The KI mice showed mild muscle weakness and characteristic histological changes in skeletal muscles, including myofibrillar disorganization, desmin-positive aggregates, p62-positive deposits, from five months of age. In conclusion, this study indicates that a dominant mutation in DNAJB4 causes adult-onset myofibrillar myopathy.