Sergio Busquets, Ivan Rakitic and Sergi Roberto joined the Argentine maestro in the UNICEF-sponsored event.
Wearing blindfolds, the blaugrana stars were shown how to kick and control the ball, which makes a noise when touched.
Unsurprisingly, Messi tucked away a spot kick as the teams drew 1-1 in a penalty shootout.
During the Paralympics, Spain lost 1-0 to China and Argentina with a game against Mexico yet to be played.