The strike left the Catalan capital's El Prat airport and main railway station almost without taxis, and follows similar protests in other major European cities, including London, last month.
A smaller strike was also expected in Madrid, but members of the city's largest taxi union did not take part and taxis were readily available in the city centre.
The strikes were the latest salvo in an ongoing dispute between taxi drivers and the providers of the new app.
Taxi drivers are concerned about the threat to their livelihoods posed by the Uber app.
But the Spanish government told strikers they will have to compete with new technologies.
Barcelona is the only Spanish city where Uber is currently in use, and the regional Catalan government has threatened drivers using it with fines of up to £5,000 and the seizure of their vehicles.
A spokeswoman for Uber said last month's protests had boosted new users in London by 850 per cent as people tried to cope with the gridlock.
The company, which is based in San Francisco and backed by Google and Goldman Sachs, has come under increasing pressure to be more transparent about its tax set-up.
Taxi associations claim Uber routes its payments through the Netherlands to minimise corporation tax payments in France, the UK and Germany - in a similar manner to Apple and Starbucks, which have found themselves under criticism.