Direct-type hyperbilirubinemia in Dubin-Johnson syndrome is due to the genetic dysfunction of multidrug resistance protein 2. However, serum bilirubin levels may fluctuate as a result of acquired conditions. Iron-reduction therapy by venesection, an alternative to interferon, was performed in a 55-year-old male patient with Dubin-Johnson syndrome complicated by hepatitis C virus-positive chronic liver disease and hepatic iron overload. His pretreatment serum total bilirubin was 10.2 mg/dl, with a dominant direct fraction. The treatment induced a significant reduction in serum total bilirubin, although it remained as high as 7.9 mg/dl. A negative correlation between serum total bilirubin and cumulative bled volume suggested that venesection could suppress bilirubin production from aged erythrocytes. The hepatic iron overload was distributed in hepatocyte lysosomes with Dubin-Johnson granules; thus, it seems that iron removal from the lysosomal granules may also help to reduce serum bilirubin. In conclusion, deep jaundice in a patient with Dubin-Johnson syndrome complicated by hepatitis C virus-positive chronic liver disease and iron overload was partially improved by iron-reduction therapy.