The Diamond Jubilee track, co-written by Lord Andrew Lloyd Webber and featuring The Military Wives among the Commonwealth Band, climbed ten spots to this week's summit.
'Sing' sold 142,000 copies to claim 2012's biggest-selling weekly number one, beating DJ Fresh's 'Hot Right Now' which sold 128,000 to reach number one in February.
Barlow said of his first number one away from Take That since 1997: "It's been such a great week, it would take something very special to top it. I'd like to say thanks on behalf of about 200 people as we've got a massive cast on this record.
"I love collaborating with other artists and being able to put them all on one stage the other night was such an honour for me. It's something I'll never forget."
'Sing' also outsold the rest of this week's top three combined, while also achieving a chart double for Barlow, who is also number one in the UK album chart.
Flo Rida's latest single 'Whistle' lands in the runner-up spot, despite only being released halfway through the past week. fun. fall one spot to three with 'We Are Young'.
Rudimental's 'Feel the Love' falls three places from last week's number one to four, while Usher's latest single 'Scream' lands at five.
DJ Fresh claims a new top ten hit with his Dizzee Rascal collaboration 'The Power' entering at six.
Rihanna features in the Top 10 twice, with her Coldplay collaboration 'Princess of China' climbing to eight ,while 'Where Have You Been' falls three to nine.
Nelly Furtado's brand new single 'Big Hoops (Bigger the Better)' is a new entry at 14, while Dot Rotten lands at 15 with 'Overload'.
Lady Antebellum's big US 2010 hit 'Need You Now' re-enters at 22, thanks to Gary Barlow and Cheryl Cole's cover at the Diamond Jubilee Concert.
Justin Bieber's latest single 'All Around the World' with Ludacris is a new entry at 30. Aiden Grimshaw's debut single 'Is This Love' enters at 35.
Ed Sheeran's 'Small Bump' also achieves a new peak of 25.
A cover version of Flo Rida's 'Whistle' by a group called Can You Blow My enters at 38, which prompted the rush-release of the original track.
Source: Official Charts Company
> This week's albums chart
Watch the video for Gary Barlow's 'Sing' below: