The ubiquitin-proteasome system is essential for intracellular protein degradation, but there are few studies of this system in the extracellular milieu. Recently, we reported that a 70-kDa sperm receptor, HrVC70, on the vitelline coat is ubiquitinated and then degraded by the sperm proteasome during fertilization of the ascidian, Halocynthia roretzi. Here, we investigated the mechanism of extracellular ubiquitination. The HrVC70-ubiquitinating enzyme activity was found to be released from the activated sperm during the fertilization process. This enzyme was purified from an activated sperm exudate, by chromatography on DEAE-cellulose and ubiquitin-agarose columns, and by glycerol density gradient centrifugation. The molecular mass of the enzyme was estimated to be 700 kDa. The purified enzyme requires CaCl2 and MgATP for activity, and is active in seawater. The purified enzyme preparation, but not the crude enzyme preparation, showed narrow substrate specificity to HrVC70. Moreover, ATP and ubiquitin are released from the activated sperm to the surrounding seawater during fertilization. These results indicate that ascidian sperm release a novel extracellular ubiquitinating enzyme system together with ATP and ubiquitin during penetration of the vitelline coat of the egg, which catalyzes the ubiquitination of the HrVC70, an essential component of ascidian fertilization.