Female employees earn up to 43.5% less at Barclays than men, according to gender pay gap figures it has submitted to the government.
Only 28 of the 1,154 companies that have reported the figures have a higher median hourly pay gap than Barclays.
Barclays chief executive Jes Staley said it had "more work to do" so women could progress in financial services.
However, Barclays said that it paid men and women in the same roles equally.
The 43.5% average gender pay gap reflects its investment bank division, Barclays International. For its UK retail bank, women earn 14.2% less than men on average, while the figure is 29.9% for the holding company, Barclays Services.
The bank has not produced an overall figure for the three divisions.