We report the accidental needlestick inoculation of a laboratory worker with vaccinia virus. Although the patient had previously been vaccinated against smallpox, severe lesions appeared on the fingers. Western blot and polymerase chain reaction-restriction fragment length polymorphism were used to analyze the virus recovered from the lesions. The vaccinia virus-specific immunoglobulin G levels were measured by enzyme-linked immunosorbent assay. Our study supports the need for vaccination for laboratory workers that routinely handle orthopoxvirus.