In this study, the glycoprotein (G) gene of the infectious hematopoietic necrosis virus (IHNV) isolate XJ-13 was cloned and inserted into a commercial vector pcDNA3.1 (+) to construct a nucleic acid vaccine (pIHNxj-G). Rainbow trout (5±0.5) g were immunized using this vaccine via the base of the dorsal fin at a dose of 2 μg vaccine per fish. At 4 and 30 days post-vaccination (d.p.v.), the rainbow trout were challenged by IHNV-XJ-13 at a dose of 100 TCID₅₀ by intraperitoneal injection. Expression of the gene in the head kidney and muscle from the vaccine delivery site was detected by real-time PCR at 4 and 7 d.p.v., respectively. During the following 65 days, fecal matter, water, and intestinal contents of the rainbow trout were collected at different time points to identify ampicillin-resistant bacteria. The results of the challenge test showed that the relative protection rate of the nucleic acid vaccine was higher than 90% at 4 and 30 d.p.v. The results of gene was significantly up-regulated in the head kidney and muscle from the vaccine delivery site. Neutralizing antibody titers showed that there was no neutralizing antibody (titer<20) in any of the sera at 4 d.p.v. At 30 d.p.v., neutralizing antibodies were detected from 8 of 10 serum samples, and the highest titer was 160. The ampicillin-resistant bacteria isolated in this study mainly belonged to sp., which are naturally resistant to ampicillin. However, no indicator bacteria like were isolated, and there were no significant differences in the number or the species of the isolated ampicillin-resistant bacteria between the vaccination group and the control. This study not only provided an effective IHNV DNA vaccine, but also provided supportive data for the safety evaluation of the IHNV DNA vaccine.