Fern Arable (DAKOTA FANNING) is a young girl growing up on the farm run by her parents (KEVIN ANDERSON & ESSIE DAVIS). On one stormy night, she rushes down to the barn and sees that a sow has had a litter of eleven piglets. But that's one more than the number of the mother's teats, and so Fern's dad is about to end the life of a runt that will otherwise starve to death. Horrified, Fern says she'll raise the piglet on her own, but her parents become concerned by her strong attachment to Wilbur who turns out to be her only friend.
When he gets too big to stay in the house, however, Fern reluctantly takes him across the road to the farm run by her uncle and aunt, Homer (GARY BASARABA) and Edith Zuckerman (SIOBHAN FALLON). There, Wilbur (voice of DOMINIC SCOTT KAY) tries to make friends with the other animals, but they'll have nothing to do with him.
There's Samuel (voice of JOHN CLEESE) the sheep who wants his flock to stop following his every move; geese Gussy (voice of OPRAH WINFREY) and Golly (voice of CEDRIC THE ENTERTAINER); Ike (voice of ROBERT REDFORD) the veteran stallion; and cows Bitsy (voice of KATHY BATES) and Betsy (voice of REBA McENTIRE) who know Wilbur's fate as a spring pig -- the slaughterhouse.
Of course, he doesn't know that, but he does find a friend in the most unlikely of animals, Charlotte A. Cavatica (voice of JULIA ROBERTS), the barn's resident spider. The other animals don't like her, but then again they don't know her, and soon she and Wilbur are the closest of friends. Also aware of Wilbur's potential fate, Charlotte tries to come up with some way to make the humans realize he should be spared, a quest that soon involves Templeton (voice of STEVE BUSCEMI), a sarcastic and self-centered rat.
He only agrees to assist if it helps him in some way. That leads to several encounters with dimwitted crows Elwyn (voice of ANDRE BENJAMIN) and Brooks (voice of THOMAS HADEN CHURCH) while he's out searching for just the right word that Charlotte can spin into her web, hoping that might save her new friend.