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.
Designer and innovator Daan Roosegaarde teamed up with Dutch civil engineering firm Heijmans to developed the technology.
The glow-in-the-dark markings are made of paint that contains "photo-luminising" powder which charges up in the daytime and slowly releases a green glow at night, 'BBC News' reported.
Once the paint has absorbed daylight it can glow for up to eight hours in the dark, doing away with the need for streetlights.
Heijmans said that the glow in the dark technology is "a sustainable alternative to places where no conventional lighting is present".
Roosegaarde's 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.
In the UK, engineers have also developed water-resistant, spray-on coating that makes roads glow in the dark by absorbing UV light during the day and releasing it at night.
The technology is being given a test run at the Christ's Pieces park in Cambridge, UK.
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