Cueva de Ardales (Málaga, Spain) is one of the richest and best-preserved Paleolithic painted caves of southwestern Europe, containing over a thousand graphic representations. Here, we study the red pigment in panel II.A.3 of "Sala de las Estrellas", dated by U-Th to the Middle Paleolithic, to determine its composition, verify its anthropogenic nature, infer the associated behaviors and discuss their implications. Using optical microscopy, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, micro-Raman spectroscopy, and X-ray diffraction we analyzed a set of samples from the panel and compared them to natural coloring materials collected from the floor and walls of the cave. The conspicuously different texture and composition of the geological samples indicatethat the pigments used in the paintings do not come from the outcrops sampled in the cave. We confirm that the paintings are not the result of natural processes and show that the composition of the paint is consistent with the artistic activity being recurrent. Our results strengthen the hypothesis that Neanderthals symbolically used these paintings and the large stalagmitic dome harboring them over an extended time span. Significance StatementThe emergence of symbolic behavior in our genus is a controversial issue. The dating of paintings in three caves from the Iberian Peninsula supports the view that Neanderthals developed a form of cave art more than 20,000 years before the emergence of anatomical modernity in Europe. In this study, we confirm that the paintings on a large speleothem from one of these sites, Cueva de Ardales, were human made, and we show that the pigments were not collected at the outcrops sampled inside the cave. Variations in the composition of the paint correspond to differences in the age of the paintings, supporting the hypothesis that Neanderthals used the speleothems symbolically over an extended time span.