The former Napoli boss, who arrived at the Stadio Olimpico in 2010, had been expected to leave the club in February, but he was eventually persuaded to remain at the helm
The 66-year-old Italian twice tendered his resignation during the Serie A season but it was rejected by the Biancocelesti board on both occasions.
However, Reja has explained that he would not take no for an answer this time around.
"My adventure at Lazio ends here," he told Cittaceleste.it. "President Lotito gave me his support and asked me to stay on.
"Even after my refusal, he invited me to reconsider and take a week to reflect. But I had already made my decision."
Reja first attempted to stand down after a home defeat by Genoa just after the start of the season, and he offered to step aside again on the eve of the Europa League semi-final second-leg clash with Atletico Madrid at the Vicente Calderon.
Reja was persuaded to continue by Lotito and the club's players both times but despite having led Lazio to a fourth-placed finish in Serie A, he has now decided to walk away.
"It seems as if we have not done anything these past two years, even though we twice qualified for the Europa League," he lamented.
"Of course, if we had hit our target of qualifying for the Champions League this year, things would have been different.
"We had several injuries, which made the season difficult, but it hurts to think that fourth place seems to be worthless.
"In addition the bid to strengthen in January went badly. After the transfer window, I found myself without five or six regulars because of drawn-out transfer negotiations which were never concluded."
Reja added that he has no idea who will be appointed as his successor, but former West Ham United boss Gianfranco Zola and Bologna coach Stefano Pioli have both previously been linked with the position.