Ben Smith, 34, began his charity attempt on 1 September 2015 and ran 284 consecutive marathons in 284 days.
He then developed an umbilical hernia in June which forced him to stop for 10 days to recover before resuming and making up the lost mileage.
Mr Smith, from Bristol, was joined by about 350 people who set off with him at 10:00 BST on his 401st marathon.
The return loop from Millennium Square in Bristol to Portishead is expected to take the runners about seven hours to complete.
Some runners will join the group from Portishead to run the half marathon distance.
Before setting off, he said he was feeling "nervous but excited" and expected the run to be emotional.
"I'm actually aching today, my legs have been alright for the last 101 days and typical, last day, legs ache, back aches, but I've got one day left," he added.
"I'm doing this to raise Â£250,000 for two anti-bullying charities, I was bullied for eight years of my life at school.
"It affected my confidence and self-esteem and that led to me trying to take my own life when I was 18."
2,528,500 calories burned
Â£163,000 raised in sponsorship
10,506 total distance, in miles
9,485 people who've run with Ben along the way
309 locations throughout the UK
101 schools visited
After the run, his team have put together a three-month "cool down" involving a month of back-to-back half marathons, followed by a month of 10km runs and a month of daily 5km runs.
Mr Smith has had to cover an extra 2.5 miles a day to make up for the time lost through injury - missing 10 days of running equates to 262 miles.
After coming out as gay, the runner wanted to raise awareness of the damage caused by bullying and support his chosen charities, Kidscape and Stonewall.
He said: "A key objective of The 401 Challenge has been to involve as many people as possible and create opportunities for children, young people and adults to discuss the difficult topics of bullying and sexuality."
By the time he finishes his last marathon he will have covered 10,506.2 miles.
Mr Smith's challenge took in 309 locations throughout the UK, with the vast majority of his marathons being distance runs organised by local running clubs.
A runner who battled a back injury is set to complete his challenge of running 401 marathons in 401 days.