A 68-year-old man with persistent bacteremia accompanying a large iliopsoas abscess, vertebral osteomyelitis, discitis and central venous port infection caused by methicillin-resistant Staphylococcus aureus (MRSA) was admitted to our hospital. During the course of treatment, the emergence of a daptomycin (DAP)-resistant MRSA strain was confirmed; the minimum inhibitory concentration was 1 to 2 μg/mL for vancomycin and more than 1 μg/mL for DAP. Although the bacterial cell wall was not significantly thickened, an increased positive surface charge and single-nucleotide polymorphism within mprF have been confirmed in DAP-resistant strains. Still rare, but clinicians need to be cautious of the emergence of DAP-resistant MRSA during treatment.