Rita Cullen passed away on Monday night at her home in Cape Cod in the US.
Their brother Aidan died just last month after a suspected heart attack.
Rita's death comes just days after Bill (70) saw the end of his car dealership empire.
The death is a big loss for the well-known businessman, who spoke affectionately about his younger sister in his best-selling autobiography, Its A Long Way From Penny Apples.
During the sixties, Rita worked for a construction company which looked after maintenance at the Kennedy family compound in Hyannis Port, Cape Cod, Massachusetts.
Another huge upset in Bill Cullen's professional life came this week as the contents of his last two remaining Renault garages were sold.
The garages, both in Dublin, were all that was left standing from a once nationwide network of Renault dealers operated by the self-made millionaire.
But after 55 years, every car, van, tool and display case in the dealerships were sold off and 45 employees at the businesses were made redundant last month.
Around 300 people turned up for the sale, with auctioneer Ricky Wilson reminding them that everything had to go.
Cullen's show room at Liffey Valley has now been put up for sale and Agents CB Richard Ellis have set a price in the region of €3.5m for a private sale.