Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation-promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation-promoting factor in Lactobacillus plantarum. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in Lactococcus lactis. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in Lb. plantarum.