The reclusive communist country topped the ministry's annual list of 50 countries where the most severe persecution of Christians has occurred in the past year.
Open Doors said the North Korean government was arresting and killing Christians, as well as torturing them in "horrible ways". The ministry has received evidence that arrested Christians are being used as guinea pigs in the testing of biological or chemical weapons.
Carl Moeller, head of Open Doors USA, said, "There is no other country in the world where Christians are being persecuted in such a horrible and systematic manner."
The report said that in spite of the hardships, Christianity was growing in North Korea and opportunities to hear the Gospel were growing, particularly in cities close to the Chinese border.
North Korea was followed on the watchlist by Iran and Saudi Arabia. Open Doors said Iran was ranked second as a result of a wave of arrests of Christians that began in 2008 and intensified in 2009. At least 85 Christians were arrested in the Islamic country, with many of them reporting mistreatment at the hands of the authorities.
Open Doors believes the arrests were a ploy of the Iranian government to distract attention away from internal unrest and to show it was still in control. The majority of those arrested have been released, although for some, court cases are still pending.
Although Saudi Arabia moved down one place on the watchlist to rank third, Open Doors said this was not because the situation for Christians had improved, but rather because there were no reports of Christians being killed or physically harmed for their faith, and only one Christian was known to have been arrested.
It remains in the top three, however, because of its strict adherence to Wahabbi Islam, which allows citizens only to adhere to Islam and deems apostasy punishable by death. Christians may worship in private, but anyone found to publicly practising their faith faces arrest, imprisonment, lashing, deportation, and torture.
Also making it into the top ten were Somalia, the Maldives, Afghanistan, Yemen, Mauritania, Laos and Uzbekistan.
Kazakhstan came off the list altogether this year, but Open Doors stressed this was not because religious freedom had improved there but because the situation for Christians in other countries had worsened.
The report noted: "This may change again if Kazakhstan implements tougher legislation on religion in the future."