AbstractSevere acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection leads to the accumulation of lipid droplets (LD), the central hubs of the lipid metabolism, in vitro or in type II pneumocytes and monocytes from coronavirus disease 19 (COVID‐19) patients and blockage of LD formation by specific inhibitors impedes SARS‐CoV‐2 replication. Here, we showed that ORF3a is necessary and sufficient to trigger LD accumulation during SARS‐CoV‐2 infection, leading to efficient virus replication. Although highly mutated during evolution, ORF3a‐mediated LD modulation is conserved in most SARS‐CoV‐2 variants except the Beta strain and is a major difference between SARS‐CoV and SARS‐CoV‐2 that depends on the genetic variations on the amino acid position 171, 193, and 219 of ORF3a. Importantly, T223I substitution in recent Omicron strains (BA.2‐BF.8) impairs ORF3a‐Vps39 association and LD accumulation, leading to less efficient replication and potentially contributing to lower pathogenesis of the Omicron strains. Our work characterized how SARS‐CoV‐2 modulates cellular lipid homeostasis to benefit its replication during virus evolution, making ORF3a‐LD axis a promising drug target for the treatment of COVID‐19.