We investigated the subchronic immunotoxicity of the phosphoramidothioate organophosphorous insecticide, acephate in white leghorn cockerels (WLH). The cockerels were divided into five groups; C1 (plain control), C2 (vehicle control), T1, T2, and T3 which received acephate suspended groundnut oil for 60 days at doses of 21.3, 28.4 and 42.6 mgkg− 1respectively. The live body weight gain, absolute and relative weights of the spleen, thymus, and bursa of Fabricius, hemoglobin (Hb), total erythrocyte counts (TEC), packed cell volume (PCV) and lymphocytes were significantly decreased. However, monocytes, eosinophils, heterophils, and basophils were significantly increased. Total protein, albumin and albumin to globin ratio, the antibody response to RD-F and delayed-type hypersensitivity response to DNCB dye or PHA-P, erythrocyte and brain Acetylcholinesterase activity was also significantly reduced in T2 and T3. At 40 and 60 days of acephate exposure, nitrate and nitric oxide production by RD-F and mitogen Con A stimulated peripheral blood and splenic lymphocytes, as well as lymphocyte proliferation in response to antigen RD-F and mitogen Con A stimulation, were significantly decreased in groups T2 and T3. Furthermore, dose-dependent increases in the frequency of micronuclei formation, varying intensity serum protein bands with different protein fractions (14.85KDa), and splenic DNA laddering (180 bp) were observed in groups T2 and T3. Histopathologically, the spleen and bursa showed morphological changes and mild lymphocyte depletion. In conclusion, low-level acephate exposure may affect acetylcholinesterase, lymphocytes, and immune responses in cockerels. As a result, it should be considered when assessing immunotoxicity and the risk to human and animal health.