On a recent episode of The Rosie Show, O'Donnell and guest Chelsea Handler had a candid discussion, during which Handler talked about getting an abortion at 16 years old.
O'Donnell later went on to ask the comedienne if she would ever sleep with a little person, prefacing the question by saying: "I'm a little ashamed about it... [but] I have a mild fear or anxiety around little people."
Amy and Matt Roloff, who star on the TLC reality show Little People, Big World, have expressed dismay at the remarks.
Amy Roloff told The Huffington Post: "I thought that was a little over the edge. I was sad and disappointed that someone like Rosie would say that. For me it's okay to have those personal feelings, but to air it as she did, I think, was inappropriate. I'm a person, I'm an adult."
The Rosie Show, which premiered on the Oprah Winfrey Network last year, has been facing low ratings and unfavourable reviews.
O'Donnell has yet to issue a response to the Roloffs' complaints.
Watch a clip from The Rosie Show below: