Despite the recent identification of surface markers of undifferentiated human embryonic stem cells (hESCs), the crucial cell-surface molecules that regulate the self-renewal capacity of hESCs remain largely undefined. Here, we generated monoclonal antibodies (MAbs) that specifically bind to undifferentiated hESCs but not to mouse embryonic stem cells. Among these antibodies, we selected a novel MAb, 4-63, and identified its target antigen as the L1 cell adhesion molecule (L1CAM) isoform 2. Notably, L1CAM expressed in hESCs lacked the neuron-specific YEGHH and RSLE peptides encoded by exons 2 and 27, respectively. L1CAM colocalized with hESC-specific cell-surface markers, and its expression was markedly downregulated on differentiation. Stable L1CAM depletion markedly decreased hESC proliferation, whereas L1CAM overexpression increased proliferation. In addition, the expression of octamer-binding transcription factor 4, Nanog, sex-determining region Y–box 2, and stage-specific embryonic antigen (SSEA)-3 was markedly downregulated, whereas lineage-specific markers and SSEA-1 were upregulated in L1CAM-depleted hESCs. Interestingly, the actions of L1CAM in regulating the proliferation and differentiation of hESCs were exerted predominantly through the fibroblast growth factor receptor 1 signaling pathway. Taken together, our results suggest that L1CAM is a novel cell-surface molecule that plays an important role in the maintenance of self-renewal and pluripotency in hESCs. Stem Cells 2011;29:2094–2099.