Abstract: With the exponential expansion of sequenced complete streptococcal genomes, numerous integrative and conjugative elements (ICEs) have been identified. However, the characteristics of ICEs in Streptococci spp. of aquatic animal origin remain enigmatic. This study aims to analyze the bioinformatic characteristics of ICEs originating from aquatic animals, verify their horizontal transfer capabilities, investigate the prevalence in bacteria, predict their microbial host distribution for horizontal diffusion, and detect their contributions to pathogenicity of host bacteria. Integrated genomic approaches were employed to systematically characterize the structural-functional attributes and epidemiological distribution of ICEs, complemented by in vitro functional assays to validate their horizontal gene transfer capacity and molecular mechanisms. A complete ICE element was identified in strain WC1535 isolated from Oreochromis niloticus, named ICE_Sag1535_mutT (abbreviation as ICESag1535). The bioinformatic characteristics of ICEs were analyzed, including their conjugation modules, recombinase composition, variable region function, and ICE homology. ICESag1535 belongs to the ICESa2603 family-like ICEs, with a length of approximately 74.1 kb, encoding 73 genes. It contains 25 core mobile genes of the Conj_Tn5252 superfamily that form the backbone of conjugant modules. The conserved backbone region of ICESag1535 includes 5 foreign gene insertion hotspots (HS), and a variable region (VR) containing 7 transposable insertion elements (IS), forming 3 complex transposons. These HS and VR can be divided into 7 major functional regions involved in the ICE functions, such as regulation of ICE stability, conjugation coupling, material transmembrane transportation, stress regulation, bacteriocin synthesis and export, integrative and conjugative evolution, host adhesion. Additionally, the 3'-terminal of ICESag1535 consists of three structurally similar tandem site-specific serine integrases (TSPSI). Cluster analysis of TSPSI revealed that S. agalactiae isolated from Oreochromis niloticus, Lithobates catesbeiana and Lates calcarifer share identical TSPSIs within ICESa2603-like ICEs distributed in the Northern hemisphere aquatic ecosystem. Homology analysis of ICEs indicated that the conserved conjugational transfer backbone of ICESa2603-like ICEs, encoded by human, pig, and fish isolates, is homologous to Conj_Tn5252 conjugant modules and has the ability to spread across species. The prevalence of ICESa2603-like ICEs in S. agalactiae from Oreochromis niloticus in Guangdong province was detected by PCR. The rate of ICE-carrying bacteria increased from 78.8% in isolates from 2014 to 2016 to 96.3% isolated from 2020 to 2021, indicating an upward trend. Conjugation experiments verified that ICE Sag1535 can excise itself from the genome, cyclize, self-transfer, and inserted into the recipient bacterium, Sag158. The attL, attR, attB, and oriT sites were characterized. After ICE integration into Sag158, the IS30 element was detected between the TSPSI and attR sites. ICESag1535, identified in this study, is a complete ICE element with self-excision and cyclization activities and cross-species conjugal transfer capability. The variable region contains a variety of complex transposons, leading to the formation of multiple horizontal transfer mechanisms and increasing prevalence diversity. Due to the recruitment specialty of IS elements, the variable region of ICEs exhibits open-ended evolution, conferring the host microbe with increased potential for pathogenicity and dissemination. This research holds significant implications for microbial safety, biological evolution, and genetic resources research.