We studied expression, regulation and function of the ABC transport proteins in ecotoxicologically relevant rainbow trout (Oncorhynchus mykiss) tissues and respective cell lines. We have done a thorough database search and found 49 ABC genes in rainbow trout. We did phylogenetic and syntenic analysis of the ABCB, C, G and H subfamilies, annotated new genes and confirmed conserved syntenic relationship for most of the genes of interest. We then evaluated gene expression of toxicologically relevant ABC transporter proteins in 7 rainbow trout tissues and cell lines. In order to test protein function we utilized the multixenobiotic resistance (MXR) assay that evaluates transport activity of target ABC proteins in relation to accumulation or efflux of specific protein substrates in inhibited versus uninhibited protein state. Finally, we obtained insight into transcriptional regulation of ABC transporters in response to model ligands/activators of toxicologically relevant transcription factors. To summarize, we offer new evidence that shows that although ABC transporters are expressed in all tested trout tissues and permanent cell lines, there are notable differences in expression levels. In addition, functional activity of most assayed transporters and transcription factors’ network implicated in transporter regulation was confirmed in studied trout cell lines. The obtained data should be taken in account when using these cell lines, especially in studies directed to extrapolation of the obtained data to mammalian systems. Stable trout cell lines appear to be a suitable model for basic screening of potential interactors, but not an optimal research model for detailed, single protein oriented studies for which heterologous overexpressing cell lines should rather be developed/used.