The study was conducted to investigate the role of aflatoxin on the infectivity and transmissibility of H9N2 AI virus. The experiment was performed on 80 non-vaccinated turkeys, divided into 4 groups of 20 birds each. Group A was kept as non-infected and a non-treated negative control; Group B was inoculated intratracheally with H9N2 AI virus (1 × 10(7) EID50) at 4 weeks of age; Group C was fed on a diet containing 0.5 ppm aflatoxin from Day 1 through the entire experiment period and Group D was fed on diet containing 0.5 ppm aflatoxin as for Group C but inoculated intratracheally with H9N2 AI virus (1 × 10(7) EID50) at the fourth week of age and then mixed with naïve birds. Infected and contact birds showed clinical signs of different severity, showing the most prominent disease signs in birds of the aflatoxin + H9N2 group. All infected birds showed virus shedding, however, the pattern of virus shedding was different for birds of the aflatoxin + H9N2 group showing pronounced virus secretion. Similarly, efficient transmission of virus was observed between infected and contact birds, but more prominent virus transmission was seen in those birds inoculated and fed aflatoxin-treated diet. Moreover, significantly lower antibody titres against H9N2 AIV were observed in birds fed aflatoxin-treated diet, indicating an immunotoxic nature of aflatoxin as the reason for poor seroconversion. Similarly, decreased IFNγ mRNA expression and higher mortality (35%) suggest an immunotoxic and immunosuppressive effect of aflatoxin leading to enhanced pathogenesis of H9N2 viruses in aflatoxin-fed birds. The immunosuppressive nature of aflatoxin might delay influenza virus clearance and this may be one of the reasons for increased pathogenicity of H9N2 LPAI viruses in turkeys under field conditions.