The country star told The Sun: "My boobs are fake, my hair's fake but what is real is my voice and my heart."
The 68-year-old drew the biggest crowd of the festival when around 100,000 people flocked to the Pyramid Stage on Sunday to see her sing hits including 'Jolene' and 9 To 5'.
Fans paid homage by dressing up as the star - who took to the stage in a white rhinestone-encrusted waistcoat and matching trousers.
But many viewing her set as it was broadcast on TV questioned whether the singer was miming, with Sky newsreader Kay Burley tweeting: "Oh, Dolly is miming. How disappointing."
Parton's spokesman told The Sun: "It's ridiculous to suggest Dolly was miming. She's been performing for more than 60 years - she doesn't need to.
"This should not overshadow what was truly a great gig and accomplished performance. Dolly adored every single moment."
Parton yesterday tweeted to her three million Twitter followers: "Wow... @GlastoFest, You were incredible!!"