The two-time Masters champion had an eagle and five birdies in the British Open warmup at Loch Lomond.
'We had a great day to score low,' Mickelson said. 'There wasn't much wind and the greens were putting very well. It's a fun test of golf and it was fun to play well the first day.'
Graeme Storm, Soren Hansen and Lee Westwood also shot 65 to tie Mickelson, while Mikko Ilonen, Marcel Siem, Mark Foster and Christian Nilsson trail by a stroke.
Ernie Els (69) had four bogeys and six birdies, and was followed by Luke Donald (70) and U.S. Open champion Angel Cabrera (71).
Colin Montgomerie, who won the European Open on Sunday, birdied three of the first five holes but settled for a 69.
'Two under. I'd have taken it at the start of the day,' Montgomerie said. 'A 4-iron at the 10th went awry and an 8-iron, the distance was wrong on the 12th. Not much wrong with the shots.'
Mickelson had been troubled by a wrist injury, but showed little sign of it Thursday.
'There is still pain. Every time you make contact, it stings,' he said. 'But I know I can't damage it any further and so I'm not afraid to go for the shots.'
After starting with two pars and a bogey, Mickelson eagled the par-5 13th and followed it up with a birdie. He then birdied four of his last six holes, including an 18-foot putt at the seventh.
'I hit a cut 3-wood around the edge of the green to 15 feet and made it. That started my round,' he said of the eagle.
Jose Maria Olazabal withdrew from the Scottish Open with a knee injury lingering from last month's U.S. Open, putting him in doubt for the British Open.