The paint on the N329 road in Oss uses a photoluminescent powder that stores up light from the sun and releases it once the sun sets.
The technology is being tested on a 500-metre stretch of road. If successful, it could be rolled out on roads all over the world.
The lines were designed by artist Daan Roosegaarde, who worked with civil engineering company Heijmans to complete the project.
The project leaders made the glowing roads as an alternative to using streetlights, and as a way of using technology to create innovative energy-efficient products.
The team is also looking to expand into technology that shows weather symbols on the road in certain conditions, with glowing snowflake shapes appearing in icy conditions.