Four women will walk for more than 24 hours to try to raise money for Great Ormond Street Hospital.
Staff at Hillhouse Primary School, in Waltham Abbey, are preparing to trek from London to Brighton.
Learning support Assistants, Jo Lawrence and Jodie Ellis, and teachers, Zoe Heffer and Nikki Savill, will take on the 100k challenge on May 24.
Ms Lawrence said the hospital was chosen because it is close to the hearts of many staff at the school in Ninefields.
She said: “We have teachers here whose children were treated at Great Ormond Street.
“Everybody seems to know someone that they have helped.”
The group is aiming to raise £1,600 between them and want to complete the 66-mile challenge in 26 hours.
A bake sale was held at the school on Friday, raising £140, which includes £20 from two year five pupils who held their sale last year.