Microcystin-LR (MC-LR) is one of the most common microcystins (MCs), which are hepatotoxic and released into a water body during a period of cyanobacterial blooms. These toxicants can be accumulated in aquatic animals and transferred along the food chain and thus pose adverse effects on aquatic environment and public health. Zebrafish Abcb4 is reported to mediate the cellular efflux of ecotoxicologically relevant compounds including galaxolide, tonalide and phenanthrene; however, it remains unclear whether Abcb4 functions in the detoxification of MC-LR. Here, we demonstrated the role of zebrafish Abcb4 in cellular efflux of MC-LR. Transcripts of zebrafish abcb4 were detected in all of adult tissues examined. MC-LR was able to induce the expression of abcb4 gene and overexpression of Abcb4 significantly decreased the cytotoxicity and accumulation of MC-LR in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcb4-G1177D mutant abolished its transporter function but not substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to this cyanobacterial toxin. Moreover, ATPase activity in developing embryos can be induced by MC-LR. Thus, zebrafish Abcb4 plays crucial roles in cellular efflux of MC-LR and is a potential molecular marker for the monitoring of cyanobacteria contamination in the aquatic environment.