Branded a bigot by John, Luigi Brugnaro took to Twitter to defend his policy, insist he was no homophobe and direct some sharp jibes of his own at the British rock star.
"Absolute respect for the homosexual choices of whoever and for different living arrangements with the associated rights and freedoms. Kids have nothing to do with that," the centre-right city leader wrote on the social media site.
He went on to accuse John, who reportedly has a property in Venice, of never offering a penny to help the city in its perpetual battle with the sea.
"You insult me to support your arguments but I think all you represent is the arrogance of the rich who can do what they like," he added in another tweet.
John, who has two sons with husband David Furnish, had used his Instagram page to launch an attack on Brugnaro that was highlighted by the Italian media on Tuesday with most commentators suggesting the mayor's comments could damage the international image of his tourism-dependent city.
"Beautiful Venice is indeed sinking, but not as fast as the boorishly bigoted Brugnaro," John, 68, wrote.
Brugnaro, who has also been attacked by Italian writers and publishers over his move, also re-tweeted numerous messages of support, including some with an overtly homophobic tone, such as: "Listen, @eltonjohndotcom: we're protecting our children from people like you."
Elected in June, Brugnaro initially removed 49 books from school libraries in line with an election pledge.
Following an outcry, many of them were returned to their libraries last month.
But works offering positive portrayals of same-sex families, such as Francesca Pardi's "Piccolo Uovo" (Little Egg) and and Ophelie Texier's renowned French work "Jean a deux mamans" (Jean Has Two Mummies) remain banned.
It is not the first time John has made waves in Italy over gay rights issues.
In March he called for a boycott of fashion brand Dolce and Gabbana after designer Domenico Dolce - who is gay himself - referred to babies conceived through IVF as "synthetic".