We have shown that multidrug resistance associated protein 1 (MRP1) mediates ATP‐dependent extrusion of bilirubin, possibly limiting its potentially toxic accumulation in cells. To determine directly if Mrp1 protects cells against unconjugated bilirubin (UCB) toxicity, mouse embryo fibroblasts (MEF) were isolated from Mrp1 knockout (−/−) mice and their wild type (WT) (+/+) littermates. Compared to WT cells, cultured MEF (−/−) cells exposed to 40–140 nM unbound [H3]‐bilirubin accumulated twice as much [H3]‐bilirubin (P < 0.01). This was associated with greater, dose‐related cytotoxicity, assessed by the methylthiazoletetrazolium test, lactate dehydrogenase release and cellular ATP content. The data confirm that Mrp1 limits intracellular accumulation of UCB and thus decreases its cytotoxicity.