John Nienstadt and Lee Anthony Piche stood down voluntarily less than seven days after Pope Francis set up a tribunal to try bishops accused of allowing priests to abuse children on their watch.
In Minnesota prosecutors had charged the Archdiocese of St Paul and Minneapolis over an alleged sexual abuse cover-up.
He was accused of "turning a blind eye" in the face of repeated allegations against a priest who was later convicted of molesting two boys.
The Vatican said in a statement that both John Nienstadt, the Archbishop of St Paul and Lee Anthony Piche, deputy bishop, resigned under a special code in canon law which allows them to step down due to illness or a "grave" reason meaning they are unfit for office.
The resignations come days after the pontiff introduced a new tribunal system allowing the Vatican to hold the highest officials in the church accountable for sex abuse on their watch.
