The controversial striker was hauled off for showboating after just 30 minutes of his side's friendly with LA Galaxy on Sunday night - before having a blazing touchline row with Mancini.
The 20-year-old Italian had already put City ahead from the spot after Micah Richards had been fouled before the bizarre incident.
Balotelli was then sent clear of the home defence on the half hour but instead of attempting to score normally, he tried a complicated pivot, followed by a backheel which rolled wide.
Mancini was clearly furious and immediately took Balotelli off, before sharing a few heated words with his fellow Italian.
"I hope this is a lesson for him," Mancini told various newspapers after the match in which City won 7-6 on penalties after it ended 1-1 in normal time.
"In football you always need to be professional, always serious and in this moment he wasn't professional.
"If you are serious, you can play 90 minutes. If not, you can come and sit by me on the bench."
The striker responded with a few choice words of his own to the City chief as he made his way to the bench, indicating that he did not understand what he had done wrong.
Mancini replied, presumably pointing out how disrespectful the move had been.
Balotelli angrily took his place on the bench before throwing his water-bottle to the ground in disgust.
He did not return to the dug-out for the second half, further fuelling an already tempestuous relationship with Mancini.
"He needs to understand his behaviour has to be good in every game - not just in a final or a semi-final but every game," Mancini added.
"He knows he made a mistake. Football should always be serious and if you have a chance to score, you should score."
Balotelli's actions add to growing crime sheet since he joined City just 12 months ago, although Mancini was keen to draw a line under the incident.
"Mario is young, I want to help him and that is the end of it," he added.
"To take him off after 30 minutes is enough punishment. It won't have been easy for him but it has to be a lesson."
Asked what Balotelli had said to him, he replied: "It was in Italian. In English I would find it difficult to give you the translation. But Mario is streetwise, he knows he was not offside."
Since arriving at City last summer Balotelli has courted controversy.
He was sent off at West Brom in November, then against Dynamo Kiev in a last 16 Europa League tie which Mancini branded "stupid".
On more than one occasion the City chief accused Balotelli of not listening to him and he raised further eyebrows when he accepted the Golden Boy award and declared of the previous winners, the only one who was a "little bit" better than him was Lionel Messi.
He did not turn up for an end-of-season trophy parade - even though he was named man-of-the-match as City defeated Stoke in the FA Cup final at Wembley.
Already this season he was pictured showing off a T-shirt of questionable taste, which was condemned by the Damilola Taylor Trust.
With City due to return to England at the end of their three-match tour, Mancini will presumably let Balotelli know exactly what he thinks, although with the future of Carlos Tevez uncertain and relations with Emmanuel Adebayor and Craig Bellamy non-existent, the Blues currently have an unhappy roster of strikers ahead of Sergio Aguero's planned ?45million move from Atletico Madrid.
Source: Team Talk
Source: Team Talk