One for Arthur was the unexpected winner - only the second victory by a Scottish-trained horse, the last being in 1979.
William Hill said it took about £25m on the race but added it would most likely have to "give it all back".
A spokesman said: "It was a National to forget for us but a fairly grand one for punters in Scotland."
Rupert Adams told the BBC Scotland news website that, for every year for the past five years, William Hill has made about £5m on the Grand National.
"We had expected this year's Grand National to be a bit of a money-spinner for us," he said.
"We won't know exactly how much we will be paying out until everyone has brought their slips in and we have paid out their money, but it looks like we have just about broken even - although we have lost significantly in Scotland.
"The problem is we won't get the money back because with the Grand National most people only make that one bet a year."
Paul Petrie, McBookie.com spokesman, said: "It was a bad day for Scottish bookmakers but a great day for punters and more importantly for Scottish racing.
"It has been a long time since we have celebrated a Scottish winner and although it has cost us a fortune, we couldn't be more happy for Lucinda Russell."
One For Arthur was ridden by Derek Fox and trained by Kinross-based Lucinda Russell, whose partner and assistant is former champion jockey Peter Scudamore.
She said: "What a day. We have a fantastic team behind us and I'm just so pleased.
"He's amazing. He's improved every time. I kept thinking barring accidents, he would win the National and he has.
"Together (Peter and I) we have had good and bad times but the horses are all back in form now.
"It's brilliant for Scotland. I said it's for Scotland, but it's really for the team and the yard.
"It's out of this world."
Bookmakers across the UK are counting the cost of Saturday's Grand National after a 14-1 shot claimed victory.