Equine herpesvirus type 8 (EHV-8) is predominantly isolated from donkeys, but its biological properties and pathogenic potential remain underexplored. This study aimed to investigate the biological characteristics and pathogenicity of the EHV-8 LCDC01 isolate by examining its effects in rabbit kidney (RK-13) cells and BALB/c mice. The virus was assessed for its ability to induce viral replication, pathological changes, and alterations in pro-inflammatory responses. In vitro, the EHV-8 infection of RK-13 cells induced characteristic cytopathic effects, including cell contraction, the formation of grapevine bundle-like structures, and detachment. In vivo, mice infected with the virus exhibited no clinical signs other than weight loss. Polymerase chain reaction (PCR) analysis detected viral DNA exclusively in the lungs of infected mice, while TaqMan PCR further confirmed the presence of EHV-8 nucleic acids in the lungs, liver, brain, and intestines. Furthermore, ELISA assays revealed a significant increase in the secretion of pro-inflammatory cytokines, including IL-1 beta, IL-6, IL-8, and IFN-alpha, in the lungs (p < 0.05). These findings suggest that EHV-8 primarily replicates in the lung tissue of mice and can induce inflammatory responses. This study provides valuable insights into the pathogenic mechanisms of EHV-8 and lays the groundwork for further investigation into its potential impact on equine and other animal populations.