In a statement released last night, the Forrests said they had decided to live apart after 31 years of marriage.
Nicola Forrest is understood to have been based in Sydney in recent months, while Andrew Forrest remained in Perth.
"Our friendship and commitment to our family remains strong," the pair said.
"There is no impact on the operations, control or direction of Fortescue, Minderoo or Tattarang.
"We will continue our shared mission to create and gift our wealth to tackle community and global challenges, as recently shown by last month's donation of one-fifth of our Fortescue shareholding to Minderoo Foundation."
In 2013, the couple signed up to the Giving Pledge in a vow to donate their wealth to charity over their lifetimes.
"Accumulating wealth should only be a small part of a person. Their contribution to their family and society is way more important. Other skills such as carpentry, farming, the arts, working in construction, or for government are equally as important. If you happen to be good at accumulating wealth, then I believe in using that skill for the greater good," Andrew Forrest said last month.
"This is why we will continue to donate our wealth to causes where we can make a sustainable difference."
Andrew Forrest is Australia's second-richest person behind fellow iron ore developer Gina Rinehart. According to Bloomberg, he is the world's 72nd-richest person.
The company he founded, Fortescue Metals Group, reiterated that the announcement would have no impact on its operations and Andrew Forrest would continue as executive chairman.
The Forrests and their companies Tattarang, Minderoo and Coaxial Ventures, the latter of which is a newly formed company controlled by Nicola Forrest, according to The Australian Financial Review, hold 36.25% of Fortescue.
At yesterday's closing price of Fortescue shares, that stake was worth A$24.76 billion.
Nicola Forrest has no direct involvement in the running of Fortescue but is heavily involved in the day-to-day at Tattarang and Minderoo.
The Forrests founded Minderoo - named after Andrew Forrest's childhood homestead - in 2001.
The foundation has donated more than US$5.9 billion to address topics including modern slavery, ocean health, cancer, Indigenous disparity, childhood development, disaster resilience, and plastic waste.
One of their three children, Grace Forrest, is a director of Minderoo and a founding director of the group's anti-slavery charity Walk Free.
Andrew and Nicola Forrest co-chair Minderoo.
The pair are also directors of Tattarang, one of Australia's largest private investment groups, which has interests in mining, energy, agriculture, food, property, health and fashion.
Tattarang's most recent investment is the A$760 million takeover of Kambalda nickel miner Mincor Resources via subsidiary Wyloo Metals.
This morning, Wyloo announced it was moving to compulsory acquisition of Mincor after last week reaching an interest of 92.7%.
