Colombia inflicted Spain's first defeat in almost a year with a 1-0 friendly victory in Lonon Friday.
Crystal Palace defender Daniel Munoz struck in the second half after a superb run by Luis Diaz to capitalise on a disappointing display from La Roja ahead of this summer's European Championship in Germany.
Spain, who won the Nations League last year to end an 11-year run without silverware after triumphing at Euro 2012, underwhelmed in their second loss under coach Luis de la Fuente.
The first came in March 2023 in a defeat by Scotland in Euro 2024 qualifying which cast early doubt over the coach's credentials for the job, with concerns easing over the subsequent months.
De la Fuente picked an experimental line-up against Colombia, handing Athletic Bilbao defender Dani Vivian his debut in the heart of defence, while resting captain Rodri among others.
On Thursday the coach said he hoped the country's football would draw admiration despite the Spanish federation's recent turmoil, making headlines this week as part of a corruption investigation that saw its headquarters searched by police.
However Spain did not shine against Nestor Lorenzo's Colombia side, who have now gone 20 matches unbeaten in a streak longer than two years.
"From defeats you learn more than from wins, it was a very hard game," said De la Fuente.
"We did some things very well but others we have to improve, especially from the second half."
La Roja did shade the first half with Vivian heading over a cross from Alejandro Grimaldo, who put in several dangerous balls, and Gerard Moreno's volley saved well by Colombia goalkeeper Camilo Vargas.
Colombian supporters made up the majority of the crowd and they were brought to their feet by Liverpool winger Diaz on occasion.
Former Real Madrid playmaker James Rodriguez came on at halftime for Colombia to further excite their fans and he turned the tide in Los Cafeteros' favour.
'Still growing'
Alex Remiro tipped over one Rodriguez strike and another sailed over, with the South American side now on top.
They took the lead after 61 minutes when Diaz exploded down the left flank and crossed to the back post where Munoz finished with an acrobatic effort.
Tottenham defender Pedro Porro came close to levelling but volleyed into the side-netting.
De la Fuente brought on 17-year-old Barcelona defender Pau Cubarsi for his debut in the final stages, becoming the second youngest player ever to play for Spain behind teammate for club and country Lamine Yamal.
Mikel Merino hammered a late chance over the bar as Spain failed to find an equaliser. They next face Brazil on Tuesday in Madrid, at the Santiago Bernabeu in another friendly.
"They pegged us back bit by bit in the second half (but) their goal came from a great individual move," said Moreno.
"We have to work, analyse ourselves, improve."
Colombia face Romania on Tuesday as they continue their warm-up for this summer's Copa America.
"In the second half we set out to show we wanted to play," said Diaz.
"We are building well for what is to come ... we're still growing."