Aflatoxin M(1) (AFM(1)) is a hydroxylated metabolite of aflatoxin B(1) (AFB(1)). After it is formed, it is secreted in the milk of mammals. Despite the potential risk of human exposure to AFM(1), data reported in literature on the metabolism, toxicity, and bioavailability of this molecule are limited and out of date. The aim of the present research was to study the absorption profile of AFM(1) and possible damage to tight junctions (TJ) of the intestinal Caco-2/TC7 clone grown on microporous filter supports. These inserts allowed for the separation of the apical and basolateral compartments which correspond to the in vivo lumen and the interstitial space/vascular systems of intestinal mucosa respectively. In this study, the Caco-2/TC7 cells were treated with different AFM(1) concentrations (10-10,000 ng/kg) for short (40 min) and long periods of time (48 h). The AFM(1) influx/efflux transport and effects on TJ were evaluated by measuring trans-epithelial electrical resistance and observing TJ protein (Zonula occludens-1 and occludin) localization. The results showed that: (i) when introduced to the apical and basolateral compartments, AFM(1) was poorly absorbed by the Caco-2/TC7 cells but its transport across the cell monolayer occurred very quickly (P(app) value of 105.10 ± 7.98 cm/s × 10(-6)). (ii) The integrity of TJ was not permanently compromised after exposure to the mycotoxin. Viability impairment or barrier damage did not occur either. The present results contribute to the evaluation of human risk exposure to AFM(1), although the AFM(1) transport mechanism need to be clarified.