Tilapia Parvovirus (TiPV) is a rising pathogen responsible for high mortality in tilapia aquaculture. Understanding TiPV’s pathogenesis is crucial for developing effective management strategies. This study aimed to elucidate TiPV pathogenesis by evaluating its cytotoxic effects on Danio rerio gill (DRG) cell monolayers and its impact on host immune responses. PCR-confirmed TiPV-infected DRG cell monolayers were subjected to an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay at 24, 48, 72, and 96 h post-infection to assess cell viability and cytotoxicity. The MTT assay revealed a progressive decline in DRG cell viability over time, with viable cell percentages decreasing from 66.71% at 24 h to 31.28% at 96 h in TiPV-infected cultures, compared to consistently high viability in controls. Simultaneously, quantitative real-time PCR (qPCR) was used to assess the expression of key immune-related genes, including Interleukins (IL-1β, IL-8), Toll-like receptor 7 (TLR7), Major Histocompatibility Complex II (MHC-II), Tumor Necrosis Factor α (TNF-α), Nuclear Factor Kappa B (NF-κB), and Chemokine Receptors (CRs).qPCR analysis showed an upregulation of IL-8, IL-1β, TNF-α, and CRs, indicating an early inflammatory response. However, significant downregulation of TLR7, MHC-II, and NF-κB suggests TiPV’s ability to modulate host immune responses. The results highlight that TiPV induces significant cytotoxicity in DRG cells, leading to severe cellular damage. The virus also alters host immune responses by modulating the expression of key immune genes, which may contribute to its virulence and persistence. These findings enhance our understanding of TiPV pathogenesis and highlight the need for targeted research to develop effective control strategies for TiPV in aquaculture systems.