The origin of eukaryotic ubiquitin-conjugating enzymes (E2s) can be traced back to the Guillardia theta nucleomorph about 2500 million years ago (Mya). E2s are largely vertically inherited over eukaryotic evolution [Lespinet, O., Wolf, Y.I., Koonin, E.V., Aravind, L., 2002. The role of lineage-specific gene family expansion in the evolution of eukaryotes. Genome Res. 1048-1059], while mammal E2s experienced evolution of multigene families by gene duplications which have been accompanied by the increase in the species complexity. Because of alternatively splicing, primate-specific expansions of E2s happened once again at a transcriptional level. Both of them resulted in increasing genomic complexity and diversity of primate E2 proteomic function. The evolutionary processes of human E2 gene structure during expansions were accompanied by exon duplication and exonization of intronic sequences. Exonizations of Transposable Elements (TEs) in UBE2D3, UBE2L3 and UBE2V1 genes from primates indicate that exaptation of TEs also plays important roles in the structural innovation of primate-specific E2s and may create alternative splicing isoforms at a transcriptional level. Estimates for the ratio of dN/dS suggest that a strong purifying selection had acted upon protein-coding sequences of their orthologous UBE2D2, UBE2A, UBE2N, UBE2I and Rbx1 genes from animals, plants and fungi. The similar rates of synonymous substitutions are in accordance with the neutral mutation-random drift hypothesis of molecular evolution. Systematic detection of the origin and evolution of E2s, analyzing the evolution of E2 multigene families by gene duplications and the evolutionary processes of E2s during expansions, and testing its evolutionary force using E2s from distant phylogenetic lineages may advance our distinguishing of ancestral E2s from created E2s, and reveal previously unknown relationships between E2s and metazoan complexity. Analysis of these conserved proteins provides strong support for a close relationship between social amoeba and eukaryote, choanoflagellate and metazoan, and for the central roles of social amoeba and choanoflagellate in the origin and evolution of eukaryote and metazoan. Retracing the different stages of primate E2 exonization by monitoring genomic events over 63 Myr of primate evolution will advance our understanding of how TEs dynamically modified primate transcriptome and proteome in the past, and continue to do so.