Spokeswoman Rasa Shevaldina said the plant ceased producing electricity one hour before midnight local time, according to schedule. The plant's first reactor was shut in 2004, the same year Lithuania joined the EU.
The RBMK reactor boasted a capacity of 1,320 megawatts, making it one of the largest nuclear reactors in the world.
Lithuania had been one of the two most nuclear-energy dependent nations along with France. Last year it tried to convince the EU to keep the plant open for another two to three years, but Brussels refused.
"We are keeping our word to our European partners," Energy Minister Arvydas Sekmokas said during a visit to the plant on Thursday.
In April 1986, an earlier, smaller version of the RBMK reactor at Ignalina exploded in Chernobyl, Ukraine, casting a fallout cloud over a wide swathe of Europe. It remains the world's worst civilian nuclear catastrophe.
But Lithuanians were proud of the Ignalina plant, which they claimed was reliable after hundreds of millions of euros were invested into safety and upgrades.
"We will witness an unprecedented event today as Lithuania becomes the first country in the world to abandon nuclear energy completely," said Viktor Shevaldin, the plant's chief.
Residents in Visaginas, a town of 25,000, are frustrated that Lithuania will lose the cheap energy source.
"I don't understand it. Why throw away a good thing that could still serve for years?" said Aleksei Tichomirov, a 47-year-old engineer who moved to Lithuania in the 1980s when the plant was built.
"This is my last day at work. There is no job in Visaginas for people like me," he said.
The Ignalina plant supplied over 70 percent of Lithuania's electricity needs. The Baltic nation of 3.4 million people will cover the shortfall by buying power on the open market from Estonia, Belarus, Ukraine and Russia.
By 2013, Lithuania hopes to build a new natural-gas power plant, but that would not be enough to meet its own energy needs.
Many Lithuanians are worried that they will become dependent on Russian gas supplies, which they fear may stop without warning given Russia's snap decisions in the past to shut off supplies to Ukraine.
However, Prime Minister Andrius Kubilius does not share the view.
"Lithuania could have done its homework better preparing for the closure, but it won't be left without energy next year. I believe our country, together with its Baltic neighbors, will have an energy market similar to the Nordic countries and other EU regions," he told Lithuanian Radio.