Zinc is an essential element for maintaining normal physiological function in living organisms. In this study, the urine proteome of rats before and after short-term intragastric administration of 82 mg/kg/d zinc gluconate (equivalent to 11.7 mg/kg/d zinc) was compared and analyzed. Many differential proteins have been reported to be zinc-related, such as mucin-2 (MUC-2) (14 times before compared with after gavage, p = 0.005) and transthyretin (3.9 times after gavage compared with before gavage, p = 0.0004). Biological processes enriched in differential proteins (e.g., regulation of apoptosis process, immune system process, etc.), molecular functions (e.g., calcium binding, copper binding, signaling receptor activity, etc.), KEGG pathways (e.g., complement and coagulation cascades, PI3K-Akt signaling pathway, etc.) showed correlation with zinc. In this study, we explore the overall effect of zinc on the body from the perspective of urine proteomics, which is helpful to deeply understand the biological function of zinc and broaden the application potential of urine proteomics.