The escalated use of copper oxide nanoparticles (CuO NPs) in various fields increased risk of human and environmental exposure. Spleen accounts for a crucial role in both innate and adaptive immunity and represents a potential target for nanoparticles toxicity. The aim of this study was to investigate the immunotoxic effect of sub-acute oral exposure to CuO NPs on spleen of adult male albino rats. Thirty rats were divided into two main groups. Group (I) control which was subdivided into two groups (10 rats each): (IA) negative control and (IB) positive control (received deionized water). Group II received CuO NPs (125 mg/kg). Animals were orally gavaged with treatments on a daily base for 28 days. At the end of experiment, blood samples were used to assess number of red blood corpuscles (RBCs), hemoglobin (Hb%), platelets (PLT), white blood cells count (WBCs), and differential leucocyte count (neutrophils, monocytes, and lymphocytes). Serum samples were analyzed for total IgG and IgM levels. Thereafter, spleens were resected to assess levels of malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF- α), and interleukin-4 (IL-4). Number of T-Lymphocyte subpopulation (CD4+, CD8+ and CD19+) and histopathology (H&E) were also determined. Results of the present study revealed that CuO NPs significantly decreased RBCs, Hb%, PLT, WBCs and lymphocytes% and significantly increased neutrophils% and monocytes%. Also, a significant reduction in total IgG and IgM in serum was detected. Furthermore, spleens of CuO NPs treated group demonstrated significant elevation in MDA, TNF- α., and CD8+. Meanwhile, SOD, IL4, CD4+, CD19+ and CD4+ /CD8+ ratio were significantly decreased. Structural damage was indicated by reduced number and cellularity of lymphoid follicles, as well as, congestion of red pulps. It can be concluded that the immune function of spleen was intoxicated by CuO NPs mostly due to oxidative stress mechanism and inflammation.