The aim of this study was to evaluate the response of ABC transporters, CYP1A and class alpha (α) GST genes, upon water and dietary exposures to benzo(a)pyrene (BaP) in Oreochromis niloticus. Partial mRNA sequences of ABC transporters (ABCB1b, ABCB11, ABCC1, ABCC2 and ABCG2) were identified, and their tissue distribution patterns evaluated in liver, gill and intestine, showing similarities with other fish and mammals. After 14 days of water exposure to BaP, ABC transporters mRNA expression was up-regulated, namely ABCC2 in gill (up to 16-fold) and ABCG2 in liver (up to 2-fold) and proximal intestine (up to 7-fold). CYP1A mRNA expression was up-regulated in water exposed animals, with maximum fold inductions of 5, 35 and 155, respectively in liver, gill and proximal intestine. After dietary exposure, intestinal CYP1A mRNA showed a 13-fold increase in exposed animals. No significant changes were seen in ABCB1b, ABCC1 and GSTα mRNA expression after both routes of exposure to BaP. In conclusion, this study has shown that transcriptional expression of some ABC transporters and CYP1A respond to the presence of BaP, indicating a possible involvement and cooperation in the detoxification process in Nile tilapia.