Abstract: P-glycoprotein is one of the major members of the ATP-binding cassette (ABC) superfamily which prevented in plasma membrane, utilizes the energy of ATP hydrolysis to transport various substrates across celluar membranes, functions as an export pump that decreases intracellular concentrations of xenobiotic agents. In our experiment, RT-PCR and RACE-PCR were used for P-gp gene cloning in swordtail fish Xiphophorus helleri. The full-length of P-gp cDNA for swordtail fish was 4301 bp with open reading frame (ORF) of 3861 bp encoding a polypeptide of 1286 amino acids. The calculated MW was of 141.64 kD and a theoretical pI of 7.06. Bioinformatics analysis indicated that the deduced amino acid sequence contained striking features of the P-gp including four core regions, two transmembrane domains (TMD) and two nucleotide-binding domains (NBD) but no signal peptide. Each TMD had six transmembrane helices while NBD contained the highly conserved motif Walker A, Walker B, ABC transporters family signature, D-loop, H-loop, Q-loop and Signature C (L-S-G-G-Q). The deduced amino acid sequence aligned with those of P-gp genes from different species also displayed high degree of sequential homology as was shown by the phylogenic tree. The gene of P-gp was expressed in all examined organs in swordtail fish and had a significantly high expression in liver, so it will be major organ for P-gp examination. Our results also showed that the hepatic P-gp expression of swordtail fish can be significantly upregulated with the exposure of benzo (a) pyrene. Therefore, the reaction of P-gp in the fish to the exposure of various xenobiotics can thus be used as a potential biomarker for monitoring environmental pollution.