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.
Turkey has inaugurated a high-speed rail link between Istanbul and Ankara, slashing by half the seven-hour journey time between the two cities.