We introduce the new vertebrate site of La Piquera (Duero Basin, central Iberian Peninsula), provide a detailed description of the lithostratigraphy and present a preliminary faunal list. The fossil vertebrate association includes amphibians (with representatives of the families Salamandridae, Alytidae, Pelodytidae, and Bufonidae), squamate reptiles (with members of the families Agamidae, Lacertidae, Anguidae, Scincidae, Blanidae, ?Erycidae, and Colubridae/Psammophiidae), and mammals (with representatives of the families Soricidae, Erinaceidae, Muridae, Cricetidae, Gerbillidae, Gliridae, Sciuridae, Vespertilionidae, Rhinolophidae, Miniopteridae, Megadermatidae, Leporidae, Prolagidae, and Bovidae). The association indicates an intermediate biostratigraphic position between the southern Iberian sites of Sifón-413 (at about 5.33–5.23 Ma) and Botardo-D (between 4.79 and 4.63 Ma), the new locality being correlated with the earliest Ruscinian (lower part of the MN14 unit). The ecological affinities of the identified small vertebrates suggest the presence of a landscape dominated by open herbaceous meadows in the surroundings of the fossil-site during the earliest Pliocene, with the occasional presence of woodland patches and stable water bodies under relatively dry and warm environmental conditions. La Piquera therefore enhances our knowledge on the vertebrate community recorded in central Iberian Peninsula during the earliest Pliocene. With more than 1200 remains, La Piquera becomes a key locality for the Early Pliocene of southwestern Europe, central Spain, where this time-span is poorly represented.