House of Lords blocks bid to give 16 and 17-year-olds EU referendum vote
In a major victory for the Government, they voted 263 to 246 against a Labour bid to lower the franchise from 18.
The result clears the way for the EU Referendum Bill to become law within days.
The Lords voted in favour of lowering the voting age last month, but that was later overturned by MPs.
During a passionate debate, Shadow Foreign Office Minister Baroness Morgan urged her fellow peers to defy the Commons again.
She said: "We believe that 16-17 year olds are and can be responsible participants in our democracy, we believe that this is their one off opportunity, a once in a generation vote on the profoundly important issue of whether we should remain a member of the EU or not.
"I urge fellow peers to support us on this issue and to give these young people the respect and voice that they deserve."
But Lord Faulks, the minister for civil justice, pointed out that the Commons had voted to keep the voting age at 18 on no fewer than five occasions.
He said: "There can be very few issues on which the elected House has express such a clear view quite so many times in the short period we have been in government.
"My Lords, we should respect that decision - repeated stand-offs between our two chambers are not good for the democratic process."
It is understood the victory was down to a number of crossbench peers deciding to vote with the Government.
A Labour source told PoliticsHome: "Seven months into the parliament, the Conservatives have finally understood how to win over the crossbenchers.
"Hopefully they will now grow up and stop whinging about Labour and Lib Dem lords ganging up to defeat them."