Streptolysin O (SLO) is a member of the cholesterol-dependent cytolysin family of toxins and a major virulence factor of group A Streptococcus (GAS) in vivo. Although cholesterol has been considered the primary receptor for SLO, SLO’s membrane binding domain also encodes a predicted carbohydrate binding site, implicating a secondary receptor. In this study, we evaluated the interaction of SLO with a potential glycosylated receptor. By flow cytometry analysis, we show that the binding and surface deposition of native toxin on primary human oropharyngeal keratinocytes can be specifically blocked by synthetic glycans lacto-N-neotetraose and lacto-N-tetraose. Inhibition of SLO-mediated pore formation by exogenous glycans significantly increased host cell viability upon toxin exposure. In order to identify host SLO-interactors, we performed a genome-wide screen using a CRISPR/Cas9 approach. Our CRISPR library screen targeting 19,050 human genes identified gRNAs for genes highly enriched after rSLO intoxication. Interestingly, these top hits were shared in a separate cytotoxicity screen of wild-type GAS infection. Focusing on the plasma membrane localization and glycosylation prediction, we selected 4 top hits as potential host receptors for SLO. To validate the screening results, we generated knockout cells by CRISPR-targeting in A549 cells. Our results indicate that knockout cells for 2 top hits are markedly more resistant to SLO-mediated cytotoxicity than WT cells at a wide range of toxin concentrations with significant reduction in pore-formation and membrane permeability. Together, these data reveal novel host proteins that play a key role in toxin recognition and control of susceptibility to SLO cytotoxicity.