The international team won by more than a minute over a pole-setting Mazda with five-time IndyCar champion Scott Dixon of New Zealand, Dutchman Renger van der Zande and Australia's Ryan Briscoe sharing the driving with Kobayashi, a former Formula One driver.
"Two times, two wins. It's amazing," said Kobayashi, who claimed the 2019 title with twice Formula One champion Fernando Alonso, Jordan Taylor and Van Der Zande in a rain-shortened race.
This time Kobayashi was behind the wheel for the closing laps.
"Wayne brought me the best car ever I drove... It's a fantastic feeling."
The team, with excellent weather conditions, completed a record 833 laps to break the previous mark of 808 set two seasons ago.
They also overcame what could have been a costly error by Briscoe, who drove through a red light on the pit lane that resulted in a penalty that dropped him from the lead to a lap down.
But Briscoe regained the lead lap under the fifth full-course caution.
NASCAR champion Kyle Busch made his debut in the race, but his team struggled in the GT Daytona class.