Bosses at Caixabank have agreed to move its base from Barcelona to the eastern city of Valencia, outside Spain's Catalonia region.
The move confirms a trend that started after Catalans voted on Sunday for secession from Spain in a poll not recognised by Madrid which in turn introduced legislation on Friday to make relocations easier.
In a statement, the bank said the reason for the relocation was to "completely safeguard the legal and regulatory framework substantial for its activity" and to remain in the eurozone and under the supervision of the European Central Bank.
Regional separatist authorities in the northeastern region of Catalonia have pledged to declare independence regardless of Spain's constitution and the opposition of central authorities in Madrid.
Caixabank's move was possible after Madrid approved a decree allowing firms' executives to bypass shareholders' approval for moving its registered address.
Half a dozen listed companies, including Banco Sabadell, have already approved a similar move.
The new decree from Madrid means Caixabank, Spain's third largest bank by assets, can now relocate before next week, when separatist authorities in Catalonia want to declare independence.
At least half a dozen other companies, including the fifth-largest lender, Banco Sabadell, have already relocated or agreed to do so.
The moves have no immediate effect on jobs or company assets, but are seen as a blow to the Catalan government.
Spain's economy minister Luis de Guindos said: "This is the result of an irresponsible policy that is causing uneasiness in the business community."
Two Catalan companies, textiles maker Dogi and reprographics company Service Point Solutions, saw their shares surge after they said they had plans to relocate.
Cava-maker Freixenet, a household name, is also considering a move while telecommunications provider Eurona and biotech firm Oryzon have already completed their relocations.