Dressed in white and accompanied by a folk band, the protesters marched in the Sinaloa state capital of Culiacan before being dispersed by some 200 police officers. One young man was injured in the head amid a shoving match.
In Mexico City, federal prosecutors filed new organised crime charges against the Sinaloa cartel leader, who has been held in the country's highest-security prison since his arrest on Saturday.
The march, which included men, women and students, began in a church on a hilltop overlooking Culiacan and culminated in front of the city's townhall amid shouts of "Viva El Chapo".
One sign held by the protesters read: "Joaquin Guzman gave jobs, not like you corrupt politicians".
A homemaker from Culiacan's blue-collar district of Las Coloradas said "El Chapo helps companies that support those in need".
"El Chapo prevents other rival cartels from entering Sinaloa," said the 30-year-old woman, who refused to give her name.
Anonymous fliers were found throughout the city earlier in the day, calling on residents to demonstrate for "the release of El Chapo, hero of Sinaloa".
Although "narcocultura" - the veneration of drug cartels through song and film - runs deep in Sinaloa, it was the first known protest in favour of Guzman in the city of 675 000 people.
Mexican marines had nearly captured Guzman in one of his Culiacan homes a week earlier, but he fled through a tunnel hidden under a bathtub that was connected to the city's drainage system.
The marines, backed by intelligence provided by the US Drug Enforcement Administration, found Guzman further south in the resort of Mazatlan.
He was staying with his beauty-queen wife and two-year-old twin daughters when he was arrested without a single shot being fired after 13 years on the run.
Federal judges ordered on Tuesday that Guzman face trial on organised crime and drug trafficking charges. US prosecutors plan to seek his extradition.
The attorney general's office announced that it was pursuing additional charges of organised crime, drug trafficking, bribery and possession of illegal weapons.
Guzman was among 12 people detained in an operation against the cartel between 11 and 22 February, the office said in a statement.
The operation netted 133 firearms, 311 cartridges, two grenade launchers, one rocket launcher, two rockets, two explosives, nine grenades, 43 vehicles (including 19 armoured cars) and 14 homes.
Authorities seized 3.1 tons of methamphetamine, 82.2kg of cocaine and 25.6kg of marijuana.