The Millers succumbed to defeat at high-flying West Bromwich Albion, meaning Millwall can afford to breathe easy going into its final two games.
At the top end of the table, the Rams claimed a potentially crucial victory over fellow play-off hopeful Bristol City.
Elsewhere, Middlesbrough kept pace with Frank Lampard's side courtesy of a come-from-behind win over lowly Reading.
MILLERS DOWN AND OUT
Having started the season among the favourites for the drop, Rotherham ultimately failed to overcome the odds.
But Paul Warne's side threatened an upset at The Hawthorns, with Clark Robertson's header handing it a lead that it held until the final 13 minutes.
The Baggies hit back with a quick-fire double through Jay Rodriguez from the spot and Rekeem Harper's strike.
That left the visitors four points behind 21st-placed Millwall, which drew 0-0 with Stoke City in the early game, with only one match left for the Yorkshire club.
Rotherham will join Ipswich Town and Bolton Wanderers, whose match with Brentford was postponed amid a player strike, in League One next season.
DERBY EDGES CLOSER
Derby headed to Ashton Gate just one point ahead of Bristol City, but a 2-0 win put it in the driving seat in the race for sixth place.
Lampard's men were on the back foot for long spells against the Robins, who fell behind to a Tom Lawrence goal.
The hosts' hopes of a turnaround were hindered by Antoine Semenyo's red card for a foul on Tom Huddlestone, with Jayden Bogle capitalising to double Derby's tally.
The Rams head to Swansea City in midweek before finishing the season at home to West Brom, while Lee Johnson's side, which is now winless in four, has Millwall and then Hull City.
PULIS KEEPS RAMS IN HIS SIGHTS
Middlesbrough is the side most likely to overtake Derby in the home straight, although Tony Pulis's men have just one game left.
Boro's 2-1 win over Reading saw it stay level with Derby on 70 points, with a goal difference that is six worse.
Lewis Wing cancelled out Danny Loader's fine finish before Britt Assombalonga netted the winner from a penalty.
The Royals had the consolation of being assured of safety courtesy of Rotherham's loss, although a far superior goal difference was always likely to keep them safe.
JOSTLING FOR POSITION
There were several games in which sides technically had nothing to play for, but that did not mean they were short on entertainment.
Preston North End and Sheffield Wednesday shared a 3-3 draw in the pick of the bunch, while Hull came from two goals down to draw 2-2 at Swansea City.
Elsewhere, Birmingham City settled for a point at home to Wigan Athletic and Nottingham Forest won 1-0 at Queens Park Rangers, which would have equalised in the 88th minute had Nahki Wells not failed to convert from the spot.