The 66-year-old twice tendered his resignation during the 2011/12 Serie A season but saw his offer to walk away rejected on each occasion.
Reja eventually guided Lazio to a fourth-place finish and qualification for the Europa League, but has made it clear that he will be vacating their managerial hot-seat with immediate effect.
"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.
"It seems as if we have not done anything these past two years, even though we twice qualified for the Europa League.
"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."
Chelsea's interim boss Roberto Di Matteo, who played for Lazio between 1993 and 1996, and former Blues and Italy striker Gianfranco Zola are the early front-runners for the vacant hotseat.