El Jueves' latest edition, which hit Spanish newsstands on Wednesday, shows several Muslims in a police lineup under the title "But … does anyone know what Muhammad looks like?"
Any depiction of the prophet is considered blasphemous by Muslims but the issue has also caused a debate in the West about censorship and freedom of speech.
The magazine declined to comment to Reuters on Thursday on the motives for the publication.
But in comments to the Huffington Post, editor Mayte Quilez said it was a decision to take a humorous position on a contentious issue.
"If you can't depict Muhammad, how do you know it is him in the cartoons?" she asked.
Last week, French satirical magazine Charlie Hebdo published cartoons of the Prophet Muhammad soon after protests against a film made in the US provoked a torrent of anti-American unrest in Egypt, Libya and other countries.
The US ambassador to Libya and three other Americans were killed in one of the first protests, on September 11, and 15 people were killed in protests in Pakistan last Friday.
The Spanish Embassy on Wednesday sent a message to its citizens in Egypt asking for caution in the event of any backlash from the El Jueves cartoon but has not said whether it will step up security in other Arab countries.
"We're still analysing what steps to take," a foreign ministry source said. – Reuters