Zabriskie, 33, who has won stages in all of cycling's grand tours and led the 2005 Tour de France for three days, completed the 29.7-kilometre flat course in 35 minutes, 59 seconds.
Germany's Jens Voigt was 23 seconds behind while American Tejay Van Garderen placed third, trailing by 34 seconds as temperatures reached 35 degrees for the second successive day.
"I came out here in February and videotaped the course," Zabriskie said. "I had a few spots where I wanted to soft pedal but maintain speed. It sounds weird, but you can do it. The turnaround was one of those spots.
"This was a really good course for me," added Zabriskie, who also won the time trial last year and finished second overall in the event in 2010.
"Any course where I don't have to get out of the aerobars is good for me. I really liked the course."
Zabriskie, who began the stage in 38th position and trailing Slovakian race leader Peter Sagan by 40 seconds, now leads Van Garderen by 34 seconds in the general classification. Robert Gesink of the Netherlands is third overall, 39 seconds behind.
Zabriskie is hopeful to be the sole U.S. time trial representative in the London Olympics.
"It's getting close to the time when they are going to have make a choice," said Zabriskie. "And I wanted to have a good performance."
Sagan, who claimed the race's first four stages in sprint finishes was 52nd on Thursday. He eased at the line, saluting race fans for his four days in the lead.
Last year's champion Chris Horner of the United States is 32nd overall, trailing by 2:50.
The eight-stage event continues on Friday with the 186.3-kilometre Palmdale to Big Bear Lake sixth stage, the first of two mountain consecutive mountain stages.
The race ends on Sunday in Los Angeles.