The current study aimed to investigate how L-thyroxine-induced hyperthyroidism and melatonin affect the immune system. The rats were divided into four groups, each with ten rats. The rats received the following treatments for four weeks: group I, control group; group II, melatonin group, treated with melatonin; group III, hyperthyroid group, treated with L-thyroxine; and group IV, hyperthyroid + melatonin group, treated with L-thyroxine and melatonin. Serum total T3 and T4 levels, CD4+ and CD8+ T lymphocytes analysis using flow cytometry, and relative gene expression of TNF-α, IL-10, IL-2, and IFN-γ were evaluated. The results revealed that hyperthyroidism substantially increased CD4+ T cells while not affecting CD8+ T cells percentage. Moreover, TNF-α, IL-2, and IFN-γ expressions were significantly upregulated in hyperthyroid rats, whereas IL-10 was not significantly altered. Melatonin administration to hyperthyroid rats significantly decreased thyroid hormone levels, increased CD4+ and CD8+ T cells percentage, upregulated IL-10, IL-2, and IFN-γ gene expression, and downregulated TNF-α gene expression. When compared to controls, the melatonin group exhibited a substantially higher percentage of CD4+ and CD8+ T cells and elevated levels of IL-10, IL-2, and IFN-γ gene expression; however, TNF-α gene expression had not changed appreciably. Therefore, it might be concluded that L-thyroxine and melatonin induce T lymphocyte proliferation and that melatonin also has anti-inflammatory properties.