SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.