"Boys have penises, girls have vaginas. Don't let them fool you. If you're born a man, you're a man, if you're a woman, you will continue to be so," read the slogan on the side of the big, orange bus, which started circulating in Madrid on Monday.
Chartered by the HazteOir ("Make yourself heard") association, the bus was part of the group's latest campaign against the promotion of gender identity, or the right for a person to feel like and be a man or a woman regardless of their birth sex.
It was bound for other Spanish cities but Madrid authorities made an official complaint over possible "LGBT-phobia," seizing the vehicle.
Prosecutors in Madrid then launched a probe into the organisation to determine whether the campaign could constitute a "hate crime", asking a judge to have the vehicle immobilised.
In a statement, they said failing to do so could risk "disturbing the peace and creating a feeling of insecurity or fear among people due to their identity or sexual orientation, and specifically among minors who could be affected by the message."
The bus campaign drew widespread criticism.
"Those from HazteOir may have penises and vaginas, but they clearly don't have a brain, nor do they have a heart," Pablo Iglesias, leader of the far-left Podemos party, said in a television interview.
Javier Maroto, a gay lawmaker for the ruling conservative Popular Party, also slammed a project he labelled a "disgraceful campaign of hate" against people who are lesbian, gay, bisexual or transgender (LGBT).
On Wednesday, the association said it was trying to get its bus back and around 20 members and supporters protested in front of Madrid's city hall.
Gerardo, a 71-year-old retiree who refused to give his surname, said he had come to defend his right to freedom of expression, just like those promoting gender identity in schools or elsewhere.
"We have the right to educate our children according to our thinking, why must they impose their criteria?" he told AFP.
"It doesn't seem right that they teach our children this type of ideology, that anyone can choose their sex."
Some 10 semi-autonomous regions in Spain have adopted laws against discrimination of transsexuals.
But activists are calling for a state-level law banning discrimination of people for their sexual orientation or gender identity.
Mane Fernandez Noriega of the State Federation of Lesbians, Gays, Transsexuals and Bisexuals, said in a statement that such a law would "protect us from these types of demonstrations regardless of where we are."
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