Background: The continuous evolution of SARS-CoV-2 has underscored the development of broad-spectrum prophylaxis. Antivirals targeting the membrane fusion process represent promising paradigms. Kaempferol (Kae), an ubiquitous plant flavonol, has been shown efficacy against various enveloped viruses. However, its potential in anti-SARS-CoV-2 invasion remains obscure. Purpose: To evaluate capabilities and mechanisms of Kae in preventing SARS-CoV-2 invasion. Methods: To avoid interference of viral replication, virus-like particles (VLPs) constructed with luciferase reporter were applied. To investigate the antiviral potency of Kae, human induced pluripotent stem cells (hiPSC)-derived alveolar epithelial cells type II (AECII) and human ACE2 (hACE2) transgenic mice were utilized as in vitro and in vivo models, respectively. Using dual split protein (DSP) assays, inhibitory activities of Kae in viral fusion were determined in Alpha, Delta and Omicron variants of SARS-CoV-2, as well as in SARS-CoV and MERS-CoV. To further reveal molecular determinants of Kae in restricting viral fusion, synthetic peptides corresponding to the conserved heptad repeat (HR) 1 and 2, involved in viral fusion, and the mutant form of HR2 were explored by circular dichroism and native polyacrylamide gel electrophoresis. Results: Kae inhibited SARS-CoV-2 invasion both in vitro and in vivo, which was mainly attributed to its suppressive effects on viral fusion, but not endocytosis, two pathways that mediate viral invasion. In accordance with the proposed model of anti-fusion prophylaxis, Kae functioned as a pan-inhibitor of viral fusion, including three emerged highly pathogenic coronaviruses, and the currently circulating Omicron BQ.1.1 and XBB.1 variants of SARS-CoV-2. Consistent with the typical target of viral fusion inhibitors, Kae interacted with HR regions of SARS-CoV-2 S2 subunits. Distinct from previous inhibitory fusion peptides which prevent the formation of six-helix bundle (6-HB) by competitively interacting with HRs, Kae deformed HR1 and directly reacted with lysine residues within HR2 region, the latter of which was considered critical for the preservation of stabilized S2 during SARS-CoV-2 invasion. Conclusions: Kae prevents SARS-CoV-2 infection by blocking membrane fusion and possesses a broad-spectrum anti-fusion ability. These findings provide valuable insights into potential benefits of Kae-containing botanical products as a complementary prophylaxis, especially during the waves of breakthrough infections and re-infections.