In fish of freshwaters environments, the accumulation and toxic effects of arsenite (AsIII) can be attenuated by detoxification proteins such as GST and ABCC transporters. We studied the effects of AsIII on the middle intestine of O. mykiss in ex-vivo and in vivo/ex vivo assays. For the ex vivo assays, we measured the transport rate of the ABCC substrate DNP-SG and GST activity in intestinal strips and everted sacs. AsIII inhibited DNP-SG transport in a concentration-dependent manner, specifically when we applied it on the basolateral side. GST activity increased when we applied a maximum concentration of AsIII. For the in vivo/ex vivo assays, we kept fish in water with or without 7.7 μmol L-1 of AsIII for 48 h. Then, we measured DNP-SG transport rate, GST activity, and PP1 activity in intestine strips during one hour. For PP1 activity, we incubated the strips with or without microcystin-LR (MCLR), a toxin excreted through ABCC2 proteins. We also analyzed Abcc2 and Gst-π mRNA expression in intestine and liver tissue. In the group exposed in vivo to AsIII, DNP-SG transport rate and GST activity were higher and the effect of MCLR over PP1 activity was attenuated. AsIII significantly induced only Abcc2 mRNA expression in both middle intestine and liver. Our results suggest that, in the middle intestine of O. mykiss, AsIII is absorbed mainly at the basolateral side of the enterocytes, excreted to the lumen by ABCC2 transporters, and is capable of modulating Abcc2 mRNA expression by a transcriptional mechanism.