Police were called to the dual carriageway in the early hours of the morning after a grey BMW travelling eastbound between Falmer and Lewes crashed shortly before 3am.
The driver, a 24-year-old man from Seaford, was killed in the collision.
Two passengers, a 24-year-old man and a 19-year-old man, both also from Seaford, were taken to hospital with serious and minor injuries respectively.
The road was closed in both directions for almost 12 hours between Falmer and Lewes while forensics teams carried out investigation work.
The A27 has since reopened in both directions.
The cause of the collision is under investigation, with anyone with information urged to email police at collision.appeal@sussex.police.uk, quoting Operation Romsley.
Officers are particularly keen to hear from anyone who may have seen the vehicle in the area around that time, or anyone who captured any relevant mobile, dash cam or CCTV footage.
A spokesman for Sussex Police said: “A man has sadly died in a single-vehicle collision on the A27.
“A grey BMW 320i was travelling eastbound between Falmer and Lewes when the incident occurred just before 3am on Tuesday 9 January.
The driver, a 24-year-old man from Seaford, was tragically pronounced deceased.
Emergency services responded to the scene and the road was closed in both directions for several hours to allow for recovery and forensics investigations, but has since fully reopened. The public are thanked for their patience and understanding.
Two male passengers in the vehicle, aged 24 and 19, and both from Seaford, were taken to hospital with serious and minor injuries respectively.
“The cause of the collision is under investigation and anyone with any information is urged to email collision.appeal@sussex.police.uk quoting Op Romsley.
“Officers are particularly keen to hear from anyone who may have seen the vehicle in the area around that time, or anyone who captured any relevant mobile, dash cam or CCTV footage.”
The closure of the A27 caused severe disruption on roads around the region as motorists sought alternative routes to get around.
Traffic came to a standstill for several hours along the A259, with reports of nine-mile queues and five-hour delays stretching from Brighton Marina to Seaford at its peak.
The B2123 between Ovingdean and Rottingdean also saw queues of around an hour, while traffic on the A27 heading eastbound stretched as far back as Hangleton.
Cars were stuck in gridlock along Ditchling Road, while delays were also reported along the B2116 from Plumpton to Ditchling and along Saddlescombe Road.
Construction work in Clayton added to the disruption as some motorists tried to get around the A27 closure.
Argus readers described the congestion as “absolute chaos” and shared stories of journeys of several hours across Brighton and the nearby area.
Commuter Tom Grant told The Argus: “I left Portslade at 7am and I got to Newhaven at 9.30am.
“In Newhaven, westbound, it was bumper-to-bumper traffic.
“Everyone was just gridlocked – it’s absolutely crazy.”
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