Local authorities covered the shape in brown paint, after it was defaced with the words “big lie”.
Victor Gonzalez, 37, was arrested on suspicion of writing the graffiti.
While some people who visited the site claimed to feel a spiritual experience, the more sceptical said the image on the wall was merely a salt stain.
Mr Gonzalez was charged with criminal damage to state-supported property.
Visitors to the site on Friday, hoping to catch a glimpse of the Madonna, said they were sorry that the image was no longer there.
Hundreds of people flocked to the motorway underpass over the past few weeks, bringing flowers and candles.
Police were ordered to patrol the emergency turnoff area under the city’s Kennedy Expressway.
The image divided observers.
“This is the first time I’ve felt something so deep in my heart,” worshipper Raven Leroux, 57, said.
But another onlooker, Victor Robles, was not convinced.
“I see just a concrete wall and an image that could happen anywhere,” he said.
The Roman Catholic Archdiocese of Chicago did not make any official endorsement of the image.