Chicken astrovirus (CAstV) is an enteric pathogen associated with white chick syndrome (WCS), a condition characterized by reduced hatchability, hypopigmentation, developmental delay, and increased early mortality. While transcriptomic studies have provided insight into host responses to CAstV infection, protein-level alterations underlying systemic pathology remain poorly defined. Here, we performed label-free quantitative proteomic profiling of the chicken spleen, a central lymphoid organ coordinating systemic immune regulation. Twenty-one-day-old SPF White Leghorn chickens were orally infected with CAstV strain PL/G059/2014 or inoculated with PBS. Spleens collected at 4 days post-infection were analyzed by nanoLC–MS/MS, and relative protein abundance was compared between infected (CA) and control (K) groups. In total, 2,529 proteins were identified (≥2 unique peptides, FDR≤1%), of which 1,118 met criteria for quantitative comparison. Among these, 33 proteins (~3%) exhibited significant differential abundance (q≤0.05), indicating a selective proteomic response to infection. Proteins with increased abundance formed a coherent type I interferon–associated antiviral module, including IFI27L1 (ISG12), OASL, STAT1, MOV10, and IFITM1. In contrast, proteins with reduced abundance were enriched for extracellular matrix components and cytoskeletal regulators, including the small leucine-rich proteoglycans decorin, lumican, and mimecan, as well as actin-associated structural proteins, indicating decreased cytoskeletal tension and stromal remodeling. Increased abundance of HSP90, together with discordant transcript–protein regulation of selected chaperones and metabolic enzymes, suggested selective modulation of proteostasis and mitochondrial metabolism. Functional enrichment and protein interaction analyses revealed two distinct response modules: a transcriptionally driven interferon-mediated antiviral program and a post-transcriptionally regulated structural and metabolic remodeling program. Integration with previously generated RNA-seq data confirmed concordant transcript–protein induction of interferon-stimulated genes, alongside transcript–protein decoupling for extracellular matrix, cytoskeletal, and mitochondrial proteins. Together, these findings demonstrate that CAstV infection induces a systemic antiviral state accompanied by stromal and metabolic reprogramming, providing a mechanistic framework linking splenic proteome remodeling to the pathogenesis of white chick syndrome.