Buruli ulcer is a chronic skin disease caused by a toxic lipid mycolactone produced by Mycobacterium ulcerans, which induces local skin tissue destruction and analgesia. However, the cytotoxicity pathway induced by mycolactone remains largely unknown. Here we investigated the mycolactone-induced cell death pathway by screening host factors using a genome-scale lenti-CRISPR mutagenesis assay in human premonocytic THP-1 cells. As a result, 884 genes were identified as candidates causing mycolactone-induced cell death, among which SEC61A1, the α-subunit of the Sec61 translocon complex, was the highest scoring. CRISPR/Cas9 genome editing of SEC61A1 in THP-1 cells suppressed mycolactone-induced endoplasmic reticulum stress, especially eIF2α phosphorylation, and caspase-dependent apoptosis. Although previous studies have reported that mycolactone targets SEC61A1 based on mutation screening and structural analysis in several cell lines, we have reconfirmed that SEC61A1 is a mycolactone target by genome-wide screening in THP-1 cells. These results shed light on the cytotoxicity of mycolactone and suggest that the inhibition of mycolactone activity or SEC61A1 downstream cascades will be a novel therapeutic modality to eliminate the harmful effects of mycolactone in addition to the 8-week antibiotic regimen of rifampicin and clarithromycin.