"We knew coming to Prague the (Czechs) would be highly motivated," he told reporters. "The players are human beings and some nights it doesn't happen. Tonight was one of those nights.
"We have to accept we weren't good enough, move forward and congratulate the Czechs on their performance," he added.
The result puts the Czech Republic level on 12 points with Group A leaders England, albeit having played a game more.
England's formation never looked right and they struggled with a lack of balance in midfield but Southgate said the problems were not just tactical.
"Whatever the balance, we didn't use the ball well enough. We have always maintained there is work to be done to be a really top team. Tonight was evidence of that. It's not a night for positives, that's for sure," he said.
England face Bulgaria in Sofia on Monday and Southgate has plenty to work on with his squad, which will be without left back Danny Rose who is suspended after picking up a yellow card.
"It's clear we have to improve without the ball. It's something we have spoken about all week," said Southgate.
"We didn't get the reaction we needed. It's going to be an interesting couple of days for us. We have to respond in the right way."
The performance was a throwback to the pre-Southgate era when England lacked fluency and it had little in common with some of their excellent attacking displays under him.
"I think we have had a lot of credit over the last couple of years and tonight we did not do enough. Our performance was not good enough, it's as simple as that. The goals we conceded were typical of the chances we gave away," he said.
Former England winger Chris Waddle, now a BBC pundit, was scathing in his assessment of the display from a team that had won all four of their Group A matches prior to Friday.
"It was very, very, very poor. Not one player played to his ability," said the former Newcastle United, Tottenham Hotspur and Olympique de Marseille player.
"They've been brilliant so far, and they have lost against a team who played well, ran their socks off and stuck to their game plan.
"I can't really see a positive tonight for England. Nobody has come off that pitch who you can say has had a good game tonight." (Reporting by Simon Evans; Editing by Ken Ferris)