Eight-year-old Jackson Sparks died on Monday after being rushed into ICU on Sunday night.
He was walking in the parade with his 12-year-old brother Tucker who was injured in the crash and is due to be discharged from hospital, according to a GoFundMe page.
“This afternoon, our dear Jackson has sadly succumbed to his injuries and passed away,” his cousin Alyssa Albro wrote.
Parents Aaron and Sheri were at their children's bedsides after Tucker sustained road rash and a fractured skull and Jackson underwent brain surgery at the Children’s Hospital of Wisconsin, according to Ms Albro.
She added: “Please know that they appreciate your continued prayers and tremendous outpouring of support for their family.
“They do however ask for privacy at this time to allow Tucker to continue to heal physically and their family to heal and mourn the tremendous loss of their sweet little boy.”
Five people ranging in age from 52 to 81 were pronounced dead within hours of the Christmas parade attack and Jackson was named as the sixth victim.
Three of those killed were members of the Dancing Grannies and had been performing at the parade.
Darrell Brooks Junior, 39, has been charged with five counts of first-degree intentional homicide, a charge that carries a mandatory life sentence if convicted.
Additional charges relating to the sixth death and the more than 60 people injured will be coming later this week or next, said Waukesha County District Attorney Susan Opper.
The criminal complaint said 62 people were injured in suburban Milwaukee, up from the 48 previously announced by police.
Brooks steered side-to-side with the intent of striking marchers and spectators, it added.
At his court hearing on Tuesday, he rocked back and forth in his seat and cried, his lawyer’s arm on his back, as the charges against him were detailed.
His bail was set at $5 million (£3.75 million) and a preliminary hearing was scheduled for January 14.
Brooks is accused of speeding away from police and entering the Waukesha Christmas parade on Sunday night, refusing to stop even as an officer banged on the roof of his SUV.
Wisconsin: Vigil for Holiday Parade victims in Waukesha
Another officer fired three shots into the vehicle, but it did not stop.
The city’s livestream video and bystander video captured the chaotic scene as the SUV sped along the parade route and then into the crowd.
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Waukesha Police Chief Dan Thompson said Brooks was leaving the scene of a domestic dispute that had taken place just minutes earlier when he drove into the parade route.
Hundreds gathered at a downtown park on Monday night in Waukesha, Wisconsin, for a candlelight vigil in honour of those lost and injured.
