The molecular basis of bilirubin toxicity to nerve cell function is still unclear. Since astrocytes are the main transporters of synaptically released glutamate and impaired glutamate uptake results in neuronal death, we investigated the effect of unconjugated bilirubin (UCB) on [³H]glutamate uptake in cultured rat astrocytes and the role of bilirubin ionization on toxicity. Astrocytes were incubated for 5–15 min, with UCB concentrations from 17 to 342 μM and UCB/albumin molar ratios of 0.2–3.0, at pH 7.0, 7.4, and 8.0. Exposure of astrocytes for 15 min to 85.5 μM UCB and 28.5 μM albumin resulted in a 63.1% decrease of glutamate uptake (p < 0.01). Interestingly, the effect demonstrated to be correlated with the UCB/albumin molar ratio (r = −0.986, p < 0.01) and a significant decrease was observed for a UCB/albumin molar ratio as low as 0.8. Inhibition of glutamate transport was also pH-dependent as it occurred at 7.4 (p < 0.05) and 8.0 (p < 0.01), but not at 7.0, suggesting that the monoanionic species of UCB accounted for the inhibition. These findings indicate that UCB, and more precisely the monoanionic species, impairs a crucial function of astrocytes such as glutamate transport and support a potential role of astrocyte function in the pathogenesis of UCB-related brain damage (kernicterus).