The outlet in High Street, Old Fletton, is to shut its doors for good on Friday (August 5).
It comes as bosses at Barclays say the number of customers using the branch has fallen to 53 who use it solely for banking.
The decision to close the branch was announced in May.
It follows the closure of Barclays’ Eastfield Road branch in January last year and its Millfield outlet that shut in December 2020.
The bank states: “The number of counter transactions has gone down in the two years to March 2020, and 91 per cent of customers also use other ways to do their banking such as online and by telephone
"Customers using other ways to do their banking has increased by 12 per cent since 2015 and in the past 12 months, 54 per cent of the branch's customers have been using nearby branches
“We’ve identified that only 53 customers use this branch exclusively for their banking.”
It prompted North West Cambridgeshire MP Shailesh Vara to write to bosses to rethink the move.
He said: “Whilst I appreciate that banking methods have changed significantly in recent years, this will still have a huge impact on some members of the local community.”
Barclays is looking to shut another 10 high street branches in the UK over October and November.
New figures show that 300 branches will shut in the UK this year including Lloyds, HSBC, NatWest and TSB.