Novel severe acute respiratory syndrome (SARS)-like coronavirus-2 (SARS-CoV-2), the cause of Coronavirus disease 2019 (COVID-19), continues to evolve into distinct lineages of enhanced transmissibility called variants of concern (VoC). The viral envelope Spike protein, originally identified as a key mediator to canonical cell entry and a primary antigen for vaccine design, harbours a significant proportion of these VoC mutations. One critical motif more recently affected by these mutations is the Arg-Gly-Asp (RGD) integrin recognition motif within the receptor binding domain (RBD) of Spike. This motif is conserved among other viruses to induce various entry signalling events in an integrin-dependent manner. In addition, Spike protein interactions can activate a diverse range of cell signalling pathways, triggering exaggerated inflammatory responses. Here, we demonstrate that the pro-inflammatory TGF-β signalling pathway is activated upon Spike protein treatment. This activity of Spike was dependent on the presence of ACE2, leading to activation of SMAD3-dependent, pro-fibrotic gene expression. Interestingly, early VoCs relied on the presence of the RGD motif to induce TGF-β signalling. However, Omicron variants beyond BA.2 do not contain this motif and subsequently have also lost this requirement. Together, our results indicate that Spike protein leads to activation of TGF-β signalling, contributing to pro-inflammatory injury and immune evasion. We propose that targeting TGF-β signalling in future vaccines and therapeutics could be crucial in reducing the continued pathogenesis of COVID-19 in all current and future SARS-CoV-2 variants.