Diego Nieto's departure, confirmed to AFP by a ministry official, comes after the culture minister Salvador del Solar resigned. The head of the country's public broadcaster has also quit.
The political turmoil follows street protests triggered by Kuczynski's December 24 pardon.
Fujimori, 79, had been imprisoned for more than a decade, serving a 25-year sentence for commanding death squads and other brutality during his 1990-2000 rule.
The pardon is being viewed by many in Peru as a reward by Kuczynski to Fujimori's family, after Fujimori's son Kenji, a lawmaker, drained votes away from an impeachment vote in Congress against the president, who is being investigated for possible corruption.
Kuczynski barely survived the impeachment vote, but the center-right leader, a former Wall Street banker, remains weakened in power, with the opposition in charge of congress.
The defense minister was absent at the swearing-in of the country's new interior minister a week ago, with his explanation that we was unwell unconvincing to some.
Nieto, a leftwing politician, had previously been culture minister.
Lawmakers had scrutinized the president for allegedly lying to cover up his ties to Odebrecht, a giant Brazilian construction company that has admitted to paying bribes to officials across Latin America to secure public works contracts.
After initial denials, Kuczynski in December admitted he had taken money from Odebrecht for what he and the Brazilian company insisted were legitimate consulting fees. He denies any wrongdoing.
The company has said it paid $20 million in kickbacks to Toledo, whom Peru wants extradited from the United States to face charges.
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