Astrocyte-neuron lactate shuttle (ANLS) signaling contributes to learning or memory and induces long-lasting nociceptive behavior. Monocarboxylate transporters (MCTs) have been reported to transport L-lactate. While MCT1, MCT2, and MCT4 are expressed in the rodent brain, it remains unclear which MCT isoform is functionally expressed by human astrocytes. Furthermore, there have been only a few reports on MCT1- or MCT4-selective inhibitors. First, we established the contribution of each MCT isoform to L-lactate transport in human astrocytes. The cellular uptake of L-lactate was found to be pH- and concentration-dependent with a Km value of 0.64 mM for L-lactate uptake. This Km value was similar to that previously established for MCT1-mediated L-lactate uptake. Next, we tried to identify a selective MCT1 or MCT4 inhibitor. While inhibitory effects were observed for MCT1 with 5-oxoproline, no inhibitory effect was observed for MCT4. Bindarit was demonstrated to exhibit selective inhibitory effect against MCT4, but not against MCT1. These findings provide novel insights into the mechanism of L-lactate transport by astrocytes, and contribute to developing novel targets for ANLS.