The musician is in negotiations to play the mother of Santana (Naya Rivera), Entertainment Weekly reports.
Estefan is due to appear in the same episode as Ricky Martin, who was linked to a role as a Spanish teacher in the show earlier this week.
"[The whole cast] is really, really big fans of hers," a source claimed. "We love her music and her husband [Emilio Estefan]'s music."
The episode is expected to feature some Estefan tracks alongside other bilingual songs.
Estefan and Martin are expected to appear in an episode of Glee in January.
The show airs on Tuesdays at 8/7c on Fox in the US and on Thursdays at 9pm on Sky1 in the UK.
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