Prime Minister Recep Tayyip Erdogan travelled aboard the first train in the service from Ankara, stopping at a town en route to make a campaign speech.
The tracks allow trains to reach speeds of up to 250 km (155 miles) per hour.
Mr Erdogan, a candidate in presidential elections in August, has pledged to overhaul Turkey's transport network.
"We made a promise. We worked hard. We crossed the mountains, and passed over rivers," Mr Erdogan said, at a campaign stop in the town of Eskisehir.
"Despite all the attempts of sabotage, blockade, and slowdown, we completed the line and opened it for service today."
The inauguration of the train link has been beset by hitches and delays, most recently when a train crashed into a maintenance vehicle.
The new service will terminate in the Istanbul suburb of Pendik, on the Asian side of the Bosphorus.
The government plans to build a further rail link, under the Bosphorus, to the centre of the city, but it is unclear when this will happen.

Small Business Saturday is in its third year, and aims to encourage people to spend locally rather than with international chains.
There are around five million small firms in the UK which employ half of the country's workforce.
The government said it wanted to help "cut red tape" for small businesses.
Business Secretary Sajid Javid said: "Small Business Saturday is an opportunity to come together and generate a real boost for small firms in the run-up to Christmas.
"Having grown up above the family shop myself, I'm proud of the UK's small businesses, providing jobs and security for millions of people and making a huge contribution to our economy."
Any firm with fewer than 50 employees is considered a small business.  In Britain 99% of all companies are in that category - employing 12.4 million people.
Last year UK traders took Â£504m on the day from an estimated 16.5 million people - a 20% increase in footfall on 2013.
But day-to-day small firms face a battle competing with big companies who can offer lower prices and have more resources to deal with issues such as administration, tax and staffing.
The Federation of Small businesses said that for every pound spent at an independent company,  around 70p stays in the local economy.

A mayday call was made at about 05:00 BST from the Pegotty, a motor cruiser taking on water just north of Grimsby dock, the Humber coastguard said.
A pilot vessel from Grimsby took two people off the boat, which sank about 25 minutes after the emergency call.
The Humber and Cleethorpes RNLI lifeboats were also launched.
The pilot vessel, Venus, and the two lifeboats returned to Grimsby after the rescue.
"The casualties were checked over and found to require no medical attention", said the RNLI.
Daniel Cooper, Cleethorpes RNLI Lifeboat operations manager, said: "Our volunteer crew managed to respond very quickly and launched the lifeboat within six minutes of the request for their assistance."
The Marine Accident Investigation Branch has been informed.
Turkey has inaugurated a high-speed rail link between Istanbul and Ankara, slashing by half the seven-hour journey time between the two cities.
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Shoppers across the UK are being encouraged to buy from independent stores on Saturday as part of a campaign to boost high streets.
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Two people have been rescued from a sinking boat in the Humber estuary after a collision with another vessel in the fog.