The men are accused of stealing the money by using a hardware device attached to a computer at the bank's Swiss Cottage branch in London after posing as engineers. Barclays Security reported a loss of the same amount in April.
The arrests follow a similar incident earlier in September, when 12 members of a gang who had attempted to take control of the bank Santander UK's computer systems were arrested, accused of trying to steal millions of pounds.
"This new and increasing methodology being seen by UK law enforcement demonstrates the rapidly evolving nature of low risk, high financial yield cyber enabled crime," the Metropolitan Police said in a statement.
The arrests, in connection with an allegation of conspiracy to steal from Barclays Bank and conspiracy to defraud British banks, were made by the Metropolitan Police's Central e-Crime Unit in London on Thursday and Friday.
Barclays were able to recover a significant amount of the stolen cash, the police said.
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