"It is with tremendous sadness that I officially hand over the Senate seat that I have held for eleven years," the Nevada Republican said in a statement.
"I stand behind my firm belief that I have not violated any law, rule, or standard of conduct of the Senate, and I have fought to prove this publicly," he said.
"(But) I will not continue to subject my family, my constituents or the Senate to any further rounds of investigation, depositions, drawn out proceedings or especially public hearings."
The 53-year-old politician cited the ongoing investigation into his handling of an affair with former aide Cynthia Hampton, whose husband Doug had been Ensign's administrative assistant.
He said the investigation had caused "simply too great" an emotional cost to him and his family.
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, a Nevada Democrat, said he "appreciated John's partnership in working with me to address our state's needs," calling Ensign a "strong advocate for Nevada."
"I know this is a difficult time for the family and I wish them all well as they work through it," he added.
Ensign's resignation, which will be effective on May 3, comes just a month and a half after he announced he would not seek reelection in 2012, saying "there are consequences to sin."
The Senate Ethics Committee is investigating claims that Ensign violated ethics rules and federal law after his affair.
Ensign acknowledged the affair in 2009, after Doug Hampton threatened to go public. The senator later confessed that his parents had paid the Hamptons $96,000 after he dismissed the couple from his payroll.
Ensign called the money a gift and the Hamptons called it severance pay. Critics say the money is an improper campaign contribution to Ensign by his parents.
A federal grand jury has meanwhile indicted Doug Hampton for violating a law forbidding congressional staff members from lobbying politicians until at least one year after they have left their Capitol Hill jobs.