In order to make a further study of the pathogenic mechanism of Acinetobacter pittii an emerging fish pathogen and provide technical reserves for the prevention and control of fish diseases caused by A. pittii, we selected A. pittii strain Ap-W20 for the whole genome sequencing and analysis of its virulence characteristics. The results showed that strain Ap-W20 contained a chromosome of 4 399 705 bp with an average G+C content of 38.78% and 4 230 coding sequences (CDS). Particularly, Ap-W20 carries four previously unreported plasmids. The GenBank accession numbers of Ap-W20 are CP027658–CP027662. The ANI analysis result confirmed that Ap-W20 belonged to A. pittii, and Ap-W20 had close similarities with A. pittii AP_882 from human (96.69%) and A. pittii PHEA-2 (96.55%) from environment. Comparative genomic analysis of Ap-W20 with AP_882 and PHEA-2 found that the numbers of gene islands, prophages and plasmids in Ap-W20 were the largest, which is likely related to their respective isolation environments and specific pathogenicity. In comparison with the VFDB database, 286 virulence genes were predicted in the entire genome of Ap-W20, mainly related to adhesion and anti-phagocytosis. Ap-W20 also has Type I, II and VI secretion systems, multiple sets of two-component regulatory system, and bacteriocin synthesis gene cluster, etc., which suggests that there may be complex pathogenic mechanisms in A. pittii derived from fish. This study demonstrates the complete genome sequence and the virulence characteristics of A. pittii from fish, which may provide a basis for the disease control caused by A. pittii.