The paint contains a "photo-luminising" powder that charges up in the daytime and slowly releases a green glow at night, doing away with the need for streetlights.
Interactive artist Daan Roosegaarde teamed up with Dutch civil engineering firm Heijmans to work on the idea.
The technology is being tested with an official launch due later this month.
It is the first time "glowing lines" technology has been piloted on the road and can be seen on the N329 in Oss, approximately 100km south east of Amsterdam.
Once the paint has absorbed daylight it can glow for up to eight hours in the dark.
Encourage innovationSpeaking to the BBC last year about his plans Mr Roosegaarde said: "The government is shutting down streetlights at night to save money, energy is becoming much more important than we could have imagined 50 years ago. This road is about safety and envisaging a more self-sustainable and more interactive world."
The team also hopes to develop giant weather symbols that appear on the roadMr Roosegaarde's projects aim to help people and technology to interact. His past projects have included a dance floor with built-in disco lights powered by dancers' foot movements, and a dress that becomes see-through when the wearer is aroused.
"I was completely amazed that we somehow spend billions on the design and R&D of cars but somehow the roads - which actually determine the way our landscape looks - are completely immune to that process," Mr Roosegaarde said.
Heijmans was already working on projects involving energy-neutral streetlights when Mr Roosegaarde teamed up with the company.
"I thought that was updating an old idea, and I forced them to look at movies of jellyfish. How does a jellyfish give light? It has no solar panel, it has no energy bill.
"And then we went back to the drawing board and came up with these paints which charge up in the daytime and give light at night," he said.
Heijmans says that the glow in the dark technology is also "a sustainable alternative to places where no conventional lighting is present".
Pilot projectInnovation on roads needs to be encouraged said Professor Pete Thomas, from Loughborough University's Transport Safety Research Centre but new technologies need to prove themselves.
"We have some high visibility markings already on roads in the UK, plus cats-eye technology etc. So the question is how much better than these is this alternative?
The Dutch Minister for Infrastructure Melanie Schultz Haegen visited the project"If we put this technology on all unlit roads that would be a lot of kilometres and it would be a big investment so if safety improvement is the target then we need hard evidence about how this compares to what we already have and to back up any safety claims," he said.
The UK Highways Agency said it was watching the trial in the Netherlands with interest but said that previous studies had shown that "luminescent road paint would be unsuitable for use in this country".
It said it would take several things in to account when deciding whether to include luminescent road markings in its design standards. These would be include how far in advance road markings could be seen, how skid resistant they were, how visible they were during the day and how they would perform in winter when there are fewer hours of daylight.
Initially the team also had plans to develop weather symbols that appeared on the road once the temperature reached a certain level. A temperature-sensitive paint mixture would be used to create giant snow flake-shaped symbols on the tarmac to warn users that the road may be icy.
The current stretch of glow in the dark road in Oss does not include this temperature sensitive technology.
It is a pilot project at this stage and is expected to expand internationally later this year. Dutch media report that Heijmans is keen to use the paint on other roads but has not yet negotiated any contracts.