Assistant head coach Jamie McAllister, the former Hearts left-back, has also departed the Stadium of Light.
The Black Cats are third in Sky Bet League One and just two points behind leaders Rotherham, but owner Kyril Louis-Dreyfus acted quickly following Saturday's demoralising result.
Louis-Dreyfus said: "I would like to thank Lee for his commitment and endeavour over the past 14 months.
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"We regret that we have had to take this decision, but felt immediate change was needed. We firmly believe that this is in the best interests of Sunderland AFC as we strive to earn promotion this season.
"Lee and Jamie will always be welcome at the Stadium of Light and they depart with our best wishes."
Johnson took charge of Sunderland in December 2020 and won 41 of his 75 games in charge, most notably the Papa John's Trophy final against Tranmere at Wembley in March 2021.
In a statement on their website, Sunderland said the process to appoint a new manager would start immediately and that the interim coaching set-up would be announced shortly.
Sunderland's next game is at home to Doncaster on Saturday.
Graham Rix signed Johnson for Hearts from Yeovil Town in January 2006 for a fee of £50,000, the midfielder signing onto a two-and-a-half-year contract.
His debut came as a late substitute in a 4–1 victory over Dunfermline, and he started his first game in another 4-1 win over Hibs.
Less than two months after Johnson joined Hearts, Rix was sacked, which resulted in Johnson receiving less game time.
He only made one substitute appearance prior to his departure six months after signing.
McAllister was already at Hearts when Johnson arrived. The former Livingston left-back was a reliable back up for Takis Fyssas and Jose Goncalves in his two and a half years at the club.
In January 2006, when Johnson arrived, McAllister agreed a two-year contract extension.
However, he was soon dissatisfied with his peripheral status at the club, particularly after the appointment of Valdas Ivanauskas as Hearts manager in April.
McAllister scored twice during his spell at Hearts.