Beckham's business group, Beckham Miami United, issued a statement saying city officials determined the proposed site was an "inappropriate location."
The statement said Beckham's group would "spend the coming months weighing alternatives" in the city.
Beckham has travelled frequently to Miami in the last few months to explore the creation of an MLS franchise and has stressed the importance of a downtown stadium location. It was the second waterfront site proposed by Beckham's group to run into local resistance.
The group headed by the former England, Manchester United and Real Madrid star needs to ink a stadium deal before it can win final approval for the franchise from MLS, the top professional league in the United States.
Scrutiny of sports stadium deals has risen sharply in Miami after $500 million in public funding was spent to build a new stadium for the Miami Marlins baseball team.
Public anger over the Marlins Park deal contributed to a 2011 recall vote that ousted former Miami-Dade County Mayor Carlos Alvarez from office.
The Miami Heat's arena deal with Miami-Dade County has also come in for public criticism as being too generous to the team, which is owned by Micky Arison, the billionaire owner of Carnival cruise line.
Beckham's group has agreed to finance a $250 million (£150m) stadium with seating for 20,000 and to pay rent on publicly owned land.
Beckham's initial preferred site for the stadium, a plot of land near the Port of Miami, faced criticism from Royal Caribbean Cruises and others who argued the stadium would interfere with port operations.
Despite the setbacks, sources close to the Beckham project said the group is committed to building a stadium in Miami. Beckham's partners include Bolivia-born cellphone services billionaire Marcelo Claure and British entertainment entrepreneur Simon Fuller.
In the statement, Beckham said the second site, sandwiched between two museums and the Heat arena, was originally suggested by the mayors of Miami and Miami-Dade County.
"Our package was the most equitable soccer stadium proposal that Miami, or any other city in America, has ever seen - 100 per cent privately funded without any local taxpayers money," the statement said.
- Sports & Recreation
- Miami Marlins
- Miami Heat
- Miami-Dade County