The cytoplasm of laticifers, which are plant cells specialized for rubber production and defense against microbes and herbivores, is a latex. Although laticifers share common functions, the protein constituents of latexes are highly variable among plant species and even among organs. In this study, transcriptomic and proteomic analyses of Euphorbia tirucalli's (Euphorbiaceae) latex were conducted to determine the molecular basis of the laticifer's functions in this plant. The hybrid de novo assembly of Illumina mRNA-seq and expressed sequence tags obtained by Sanger's sequencing revealed 26,447 unigenes. A unigene similar to Arabidopsis embryo-specific protein 3 (AT5G62200), which is a PLAT domain-containing protein, and rubber elongation factor showed the highest expression levels. The proteome analysis, studied by liquid chromatography–mass spectrometry with the de novo assembled unigenes as the database, revealed 161 proteins in the latex, 107 of which were not detected in the stem. A gene ontology analysis indicated that the laticifer's proteome was enriched with proteins related to proteolysis, phosphatase, defense against various environmental stresses and lipid metabolisms. D-mannose-binding lectin, ricin (which lacked the N-terminal conserved ribosome-inactivating protein domain), chitinase and peroxidase were highly accumulated, as confirmed by two-dimensional polyacrylamide gel electrophoresis. Thus, the lectins and chitinase may be the major defensive proteins against pests, and the other defense-related proteins and transcripts detected in latex may work in coordination with them. Highly expressing unigenes with unknown functions are candidate novel defense- or rubber production-related genes.