Pedersen, 24, clocked two minutes 19.11 seconds to beat the previous mark of 2:19.59 set by American Rebecca Soni at the Olympic Games in London last year.
The Dane, whose previous best was 2:20.53, finished fourth in London and was seventh at the last world championships in Shanghai two years ago.
"It was very fast," Pedersen said in an interview with Eurosport.
"I am really happy with the personal best and I really hope I can improve in the final (on Friday)."
Liu Zige of China won gold in the women's 200 metres butterfly.
Liu, the world record holder and third at the last edition in Shanghai two years ago, clocked a time of two minutes 04.59 seconds.
Zhao Jing of China won gold in the women's 50 metres backstroke.
Zhao, who set the world record of 27.06 seconds when she won the title in Rome in 2009, clocked 27.29 with teenage compatriot Fu Yuanhui taking silver in 27.39 and Aya Terakawa of Japan bronze in 27.53.
The 50 backstroke is a non-Olympic event.
United States won gold in the women's 4x200 metres freestyle relay.
Spain's Mireia Belmote Garcia, Olympic silver medallist in London last year, and roared on by the home crowd at the Palau Sant Jordi, claimed silver in 2:04.78 and Katinka Hosszu of Hungary bronze in 2:05.59.
Teenager Missy Franklin powered past Australia's Alicia Coutts on the final leg at the Palau Sant Jordi to lead the U.S. team home in a time of seven minutes 45.14 seconds.
Australia took silver in 7:47.08 and France, who led at the halfway stage, bronze in 7:48.43.
The US triumph, adding to their victory in Sunday's 4x100 freestyle relay, brought a fourth gold of the week for Franklin and a third for Katie Ledecky.
The result was the same as the 4x200 freestyle final at the Olympic Games in London last year.
- Sports & Recreation
- Rebecca Soni