Irwin was killed by a stingray barb to the heart on Batt Reef, off the remote resort town of Port Douglas in northeastern Queensland state, his wildlife park Australia Zoo said in a statement.
Crew members aboard Irwin's boat, Croc One, called emergency services in the nearest city, Cairns, and administered CPR as they rushed the boat to nearby Low Isle to meet a rescue helicopter. Medical staff pronounced Irwin dead a short time later, the statement said.
Irwin was famous for his enthusiasm for wildlife and his catchword "Crikey!" in his television program "Crocodile Hunter," which was first broadcast in Australia in 1992 and has aired around the world on the Discovery channel.
He rode his image into a feature film, and developed the Australia Zoo as a tourist attraction.
"The world has lost a great wildlife icon, a passionate conservationist and one of the proudest dads on the planet," John Stainton, Irwin's friend and producer, said in the statement. "He died doing what he loves best and left this world in a happy and peaceful state of mind. Crocs Rule!"
Foreign Minister Alexander Downer, who used a photograph of his family at Australia Zoo for his official Christmas card last year, hailed Irwin for his work in promoting Australia through projects such as "G'Day LA," an Australian tourism and trade promotion week in Los Angeles in January.
"The minister knew him, was fond of him and was very, very appreciative of all the work he'd done to promote Australia overseas," Downer's spokesman Tony Parkinson said.
Irwin's dedication to conservationist causes led him to become a vocal critic of wildlife hunts in Australia. The federal government recently dropped plans to allow crocodile safaris for wealthy tourists in the Northern Territory following his vehement objections.
Irwin told the Australian television program "A Current Affair" that "killing one of our beautiful animals in the name of trophy hunting will have a very negative impact on tourism, which scares the living daylights out of me."
However, Irwin had also received negative publicity in recent years. In January 2004, he stunned onlookers at the Australia Zoo reptile park by carrying his month-old son into a crocodile pen during a wildlife show. He tucked the infant under one arm while tossing the 13-foot reptile a piece of meat with the other.
Authorities declined to charge Irwin for violating safety regulations.
Later that year, he was accused of getting too close to penguins, a seal and humpback whales in Antarctica while making a documentary. Irwin denied any wrongdoing, and an Australian Environment Department investigation recommended no action be taken.
He is survived by his American wife Terri, from Eugene, Ore., and their daughter Bindi Sue, 8, and son Bob, who will turn 3 in December.
The couple met when she went on vacation in Australia in 1991 and visited Irwin's Australia Zoo; they were married six months later. Sometimes referred to as the Crocodile Huntress, she costarred on her husband's television show and in the 2002 movie, "The Crocodile Hunters: Collision Course."
Stingrays have flat bodies and tails with serrated spines, which contain venom and can cause cuts and puncture wounds. The creatures are not aggressive and injury usually occurs when a swimmer or diver accidentally steps on one.