Striker Kayode Odejayi wrote himself into the history books with a towering second-half header that sent the millionaires of Chelsea crashing out of the competition at the sixth round stage.
It was little more than the home side deserved after they had delivered a magnificent team performance that was even better than the heroic show they put on against Liverpool in the previous round.
Only Joe Cole lived up to his reputation as Chelsea were otherwise out-fought and out-thought by a fired up Championship club.
Hungarian striker Istvan Ferenczi went close to giving Barnsley the lead in the 37th minute when he got the better of John Terry but sent his fierce volley flashing just wide.
Seconds later, the outstanding Odejayi powered down the right and drilled in a low shot that was saved well by the legs of Carlo Cudicini.
Cole twice saw good efforts brilliantly blocked by Rob Kozluk as the Barnsley defence worked overtime in giving great protection to goalkeeper Luke Steele.
Having been the better side in the first half, Barnsley had to defend well early in the second as Chelsea tried to raise their game.
Just three minutes in, Cole fluffed a glorious chance after being put through by Nicolas Anelka and then the French striker himself then fired straight at Steele when well placed.
But it was Barnsley who made the breakthrough in the 66th minute when a brilliant cross from Martin Devaney saw Odejayi rise high above Cudicini to head into the net and start the delirious Barnsley celebrations.
From then on it was all about whether the home side could hold out and they did so despite frantic, late Chelsea pressure.