A Cardinals statement read: "We are devastated to learn of Jeff Gladney's passing. Our hearts go out to his family, friends and all who are mourning this tremendous loss."
"We are asking prayers for the family and privacy at this most difficult time," his agent, Brian Overstreet, added in a statement to the Fort-Worth Star-Telegram.
Gladney was involved in all 16 Minnesota Vikings games in his rookie season - starting all but one.
The cornerback recorded 81 tackles and three pass break-ups, in addition to one forced fumble.
He was released at the end of that season following an indictment for felony assault and did not play in the NFL last season.
However, after being found not guilty in a trial he signed with the Cardinals in March.
Gladney is the second active NFL player to die in a road accident this off-season, after 24-year-old Pittsburgh Steelers quarterback Dwayne Haskins was hit by a car in Florida last month.
Prior to being picked up by the Vikings in 2020, Gladney was a four-year starter at Texas Christian University.
During his four years at TCU (2015-2019), his performances saw him earn all-Big 12 Conference by Pro Football Focus by all-Big 12 coaches in 2019.
"Our TCU Athletics family, and especially our football program, was very devastated to learn of the passing of Jeff Gladney," said TCU athletic director Jeremiah Donati.
"After earning his degree and continuing his playing career in the NFL, Jeff maintained his close ties to TCU.
"He loved everything about his alma mater. He was a frequent visitor to campus and was at our spring practices and spring game this year, proudly joined by his young son."