Lactase persistence (LP) is a genetically determined trait whereby the enzyme lactase is expressed throughout adult life. Lactase is necessary for the digestion of lactose—the main carbohydrate in milk—and its production is downregulated after the weaning period in most humans and all other mammals studied. Several sources of evidence indicate that LP has evolved independently, in different parts of the world over the last 10,000 years, and has been subject to strong natural selection in dairying populations. In Europeans, LP is strongly associated with, and probably caused by, a single C to T mutation 13,910 bp upstream of the lactase ( LCT ) gene (-13,910*T). Despite a considerable body of research, the reasons why LP should provide such a strong selective advantage remain poorly understood. In this study, we examine one of the most widely cited hypotheses for selection on LP—that fresh milk consumption supplemented the poor vitamin D and calcium status of northern Europe’s early farmers (the calcium assimilation hypothesis ). We do this by testing for natural selection on -13,910*T using ancient DNA data from the skeletal remains of eight late Neolithic Iberian individuals, whom we would not expect to have poor vitamin D and calcium status because of relatively high incident UVB light levels. None of the eight samples successfully typed in the study had the derived T-allele. In addition, we reanalyze published data from French Neolithic remains to both test for population continuity and further examine the evolution of LP in the region. Using simulations that accommodate genetic drift, natural selection, uncertainty in calibrated radiocarbon dates, and sampling error, we find that natural selection is still required to explain the observed increase in allele frequency. We conclude that the calcium assimilation hypothesis is insufficient to explain the spread of LP in Europe.