Lemon entered the courthouse in Minnesota's capital St Paul wearing a black suit. On his way in, he clapped his hands and waved to supporters, but did not respond to questions from reporters.
Standing at a lectern and flanked by his lawyers, Lemon answered "not guilty" when asked for his plea.
Lemon, now an independent journalist, livestreamed a protest against Trump's deployment of thousands of armed immigration agents into Democratic-governed Minnesota's biggest cities.
The protest disrupted a January 18 service at Cities Church in St Paul. He was charged with conspiring to deprive others of their civil rights and violating a law that has been used to crack down on demonstrations at abortion clinics but also forbids obstructing access to houses of worship.
The courtroom on Friday local time was packed with people supporting Lemon and the church protesters, who were also there to be arraigned.
Dozens of Lemon supporters gathered in front of the courthouse for a protest, chanting "drop the charges" and "protect free speech".
They held signs with slogans including "Lemon was just doing his job" and "ICE out".
Following the plea, Lemon, standing next to his husband, told reporters he would fight the "baseless charges" against him and would not be silenced.
"The events before my arrest and what has happened since show that people are finally realising what this administration is all about," Lemon said.
"The process is the punishment with them, and like all of you here in Minnesota. ... I will not back down."
Lemon's lawyer has called the case an attack on First Amendment free speech rights.
The president, who has frequently criticised Lemon, praised the Justice Department for bringing the charges against the journalist, calling the disruption of the church service a "horrible thing".
Thousands of protesters took to the streets of Minneapolis and other US cities in January to denounce an immigration crackdown in which federal agents fatally shot two US citizens, Renee Good and Alex Pretti.
Trump has since agreed to end his deportation surge in Minnesota, with many agents set to return to their home states over the next week.
Organisers of the Cities Church protest told Lemon they chose the church because they believed a senior pastor there was working with US Immigration and Customs Enforcement.
In a livestream archived on his YouTube channel, Lemon can be seen meeting with and interviewing the activists before they go to the church.
He then records the disruption inside and interviews congregants, protesters and a pastor, who asks Lemon and the protesters to leave.
Lemon spent 17 years at CNN, becoming one of its most recognisable personalities, and frequently criticises Trump in his YouTube broadcasts.
Lemon was fired by CNN in 2023 after making sexist on-air comments for which he later apologised.
