In a statement, the fiery Italian cited "a number of broken promises" and claimed his position had "become untenable" with continuing uncertainty over the club's ownership.
The 44-year-old, who guided the Robins to promotion last season, claimed he had actually tendered his resignation last Tuesday but kept it under wraps until meeting with the club's prospective new owners on Friday.
As a result of those discussions, Di Canio revealed he agreed to stay on as Swindon boss only to make another U-turn with this announcement as a takeover was still yet to be rubber-stamped.
In the statement, Di Canio said: "Last Tuesday, 12th February, I formally confirmed my resignation as manager of Swindon Town Football Club with immediate effect. There have been a number of broken promises made by the club over the time that I have been manager of Swindon Town."
He went on: "I did not resign immediately, nor publicise my resignation after I had formally confirmed it as I did not want to jeopardise the negotiations for the sale of the club and I wanted to listen to what the proposed new owners' plans were.
"Following discussions, my representatives put forward a proposal that would secure my future at the club until at least the end of the season and quite possibly beyond as well.
"This proposal actually reduced the club's contractual liabilities to me in the interest of saving Swindon Town FC. At a meeting last Friday the proposed new owners accepted and said they were very happy with the new terms.
"I entered into a temporary arrangement with the proposed new owners to continue in my role as manager, but all this was agreed subject to Football League approval for the purchase of the club being granted by 5:00pm on Monday 18th February.
"At the time of issuing this statement, I have had no further contact from the proposed new owners and I have been told that unfortunately Football League approval has not been granted yet and therefore my temporary arrangement has ended and my resignation stands.
"Thanks to the Swindon Town supporters for the incredible support they have shown me and the team throughout my team as manager."
Di Canio was also unhappy that winger Matt Richie was sold for £500,000 to Bournemouth against his and the player's agent's wishes.
Swindon's board were left to rue the resignation and pledged to try and sort out their affairs in order to achieve promotion to the Championship.
Town head to Tranmere on Tuesday under the care of Fabrizio Piccareta, knowing a win would put them top of League One.
A club statement read: "The board are disappointed that Paolo Di Canio has made the decision to resign at such a critical time for Swindon Town Football Club.
"We are also disappointed that the sale of the club has yet to be approved, but we hope the outstanding issues with the Football League will be resolved within a short period of time.
"It is worth noting that Matt Ritchie was sold due to the tremendous financial pressure the club is facing and in order to continue operating whilst the sale of the club went through.
"We understand the prospective new owners had reached agreement with Paolo Di Canio on bringing in loan players to strengthen the squad for a final push for the Championship.
"The board shares the fans' tremendous frustration at the events of the last few weeks and is determined to resolve these issues to ensure the best outcome for the club."
Di Canio landed his first job as a coach when he was appointed at Swindon in May 2011.
He guided the club to the League Two title a year later and said he was prepared to eat into his savings to fulfil his ambition of taking the team into the second tier.
"I'm not going to start selling my houses but I want to keep those players," Di Canio said at the time.
"When I die I will have a bit less money but I will have the victory as a manager. I've spent big money on solicitors in the past - you can imagine what I would spend on my players."