Richard Bland and Emanuele Canonica are two strokes off the pace at 6-under- par 210 after rounds of 68 and 69, respectively. Bradley Dredge and Damien McGrane share fourth place one stroke further back, while co-overnight leader Steve Webster is tied with a group in sixth place at 4-under-par 212.
Mark Foster, who shared the overnight lead with Webster, fell into a tie for 19th place at 1-under-par 215 after a disappointing 5-over 77 in his round.
Colsaerts, who collected six birdies to go along with one bogey, was in the lead for much of the round and is in good position to claim his maiden European Tour victory.
'I went fine because I was just playing well,' said Colsaerts of the emotions involved in leading the field. 'The shots were just coming off and I really never thought about it at all.'
Colsaerts' best finish so far this season was a tie for 21st at the Jazztel Open de Espaa en Andaluca. He's not having a particularly good year, but likes his chances at The Gleneagles Hotel's PGA Centenary Course.
'I kind of always liked this place. It's a really pure track and if you hit good shots you are rewarded,' said Colsaerts, who then joked that the cool and rainy weather 'so far has been really good for Scotland.'
Colsaerts, who entered the round in a sixth-place tie at 3 under, got off to a scorching start with birdies on three of his first five holes, including the 516-yard par-5 second.
That moved the 22-year-old Belgian to 6 under par, where he would remain around the turn and until a birdie at the par-5 12th gave him a score of minus-7.
Colsaerts dropped a shot with a bogey at No. 15, but then rebounded with a birdie at the 16th and another at the 18th to finish off his round. He has only hit 22-of-39 fairways so far in the tournament, but a good approach game and a little luck has him in the lead.
'I got a lot of good lies, so that always helps,' Colsaerts said. 'I got quite lucky in the rough, actually.'
Bland, who is also seeking his first European Tour victory, opened his round by three-putting from 4 feet for a bogey at the first. But the Briton recovered with five birdies and an eagle at the par-5 12th and finished with a 4-under round.
Italian Canonica also opened his round with a bogey at No. 1, but that was his only misstep in a minus-3 round.
Dredge and McGrane both shot 69s to stay close, while Wade Ormsby and Barry Lane had the rounds of the day with 6-under 66s. Ormsby shares sixth place with Webster and Ian Garbutt. Lane is tied with three others one stroke further back.