This study was conducted to investigate the effects of aflatoxin B-1 (AFB(1)) on T-cell subsets and mRNA expression of cytokines in the small intestine of broilers. One hundred and fifty-six one-day-old healthy Cobb broilers were randomly divided into control group (0 mg/kg AFB(1)) and AFB(1) group (0.6 mg/kg AFB(1)) with three replicates per group and 26 birds per replicate for 21 days, respectively. At 7, 14, and 21 days of age, the duodenum, jejunum and ileum were sampled for analyzing T cell subsets (CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+)) by flow cytometry as well as IL-2, IL-4, IL-6, IL-10, IL-17, IFN- and TNF- mRNA expression by qRT-PCR. The percentages of T-cells in the intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of duodenum, jejunum and ileum in the AFB(1) group showed a decreased tendency in comparison to the control group. The mRNA expression of cytokines in the three intestinal segments in the AFB(1) group presented a general decline compared with the control groups. Our data demonstrated that 0.6 mg/kg AFB(1) in the broilers diet could reduce the percentages of T-cell subsets and the expression level of cytokine mRNA in the small intestine, implying that the immune function of the intestinal mucosa might be affected. The reduction of cytokines mRNA expression may be closely associated with the decreased proportions of T cells subsets induced by AFB(1).