The National Oceanographic and Atmospheric Administration's authoritative Keeling lab atop the Mauna Loa volcano in Hawaii has been measuring concentrations of the greenhouse gas since 1958.
The last time CO2 was consistently above 400ppm was about 3 million to 5 million years ago, before modern humans existed, climate scientists said.
While the levels will rise and fall considerably over time periods, the long-term trend is a steady increase, they said.
"Carbon dioxide has some variability on an hourly, daily and weekly basis, so we are not comfortable calling a single number -- the lowest we will go is on a daily average, which has happened in this case," NOAA's James Butler told the BBC.
"This is the first time the daily average has passed 400ppm at Mauna Loa."
"Probably next year, or the year after that, the average yearly reading will pass 400pm," he said.
In eight or nine years, he said, "we will probably have seen the last CO2 reading under 400ppm."
Carbon dioxide in the atmosphere is chiefly a result of the burning of fossils fuels.