The west London airport said the measure will keep passengers arriving from destinations on the high-risk list away from other travellers.
Britain's red list has been resurrected, with 10 countries in southern Africa added since Friday November 26 due to concerns about the Omicron coronavirus variant.
People entering the UK from those locations must spend 11 nights in a quarantine hotel at a cost of £2,285 ($3,042) for solo travellers.
Heathrow first opened a facility for red list arrivals at Terminal 3 on June 1, following concerns that allowing passengers to mix with those who had flown in from other locations could increase the spread of the virus.
It was later switched to Terminal 4 but was closed in early November following the removal of the final seven countries on the list. The facility was reopened at 3pm on Wednesday.
Airline bosses expressed cautious optimism on Wednesday that the spread of the Omicron variant would not wreck the industry’s recovery from the coronavirus pandemic.
Many governments have tightened border restrictions amid concerns about the variant, prolonging measures that have plagued the travel sector since the start of the pandemic.
But speaking at a World Aviation Festival in London, industry leaders said they were better prepared than before and had not seen sharp declines in bookings because of Omicron.
Tougher travel rules introduced by the government include requiring fully vaccinated people entering the UK to self-isolate until they receive a negative result from a PCR test.
Previously they were only required to take a cheaper lateral flow test and did not need to self-isolate unless their test result was positive.
Heathrow’s chief operating officer Emma Gilthorpe said: “We are supportive of measures that protect public health and prevent the spread of Covid.
“The rapid introduction of restrictions for international travel will nonetheless be a further significant blow for British exporters and those wanting to visit friends and relatives.
“Keeping the changes under constant review, and a government commitment to the removal of red list countries as soon as it is safe to do so, will help.
“Heathrow maintains the highest levels of Covid-secure measures to ensure our passengers, colleagues and partners know that Heathrow is a safe place to travel to and from.”
More than three million passengers travelled through Heathrow in October, marking the sixth consecutive month of growth.