•SARS-CoV-2 Spike protein (Spk) induces intestinal chloride secretion in mice;•Spk causes lysozymes depletion by Paneth cells degranulation;•Spk diminishes mucus blanket associated with mucin 2 reduction;•Mucosal intestinal barrier is impaired by Spk inoculation on the gut;•Spk triggers local inflammation and dysmotility on the gut. Studies have reported the occurrence of gastrointestinal (GI) symptoms, primarily diarrhea in COVID-19. However, the pathobiology regarding COVID-19 in the GI tract remains limited. This work aimed to evaluate SARS-CoV-2 Spike protein interaction with gut lumen in different experimental approaches. Here, we present a novel experimental model with the inoculation of viral protein in the murine jejunal lumen, in vitro approach with human enterocytes and molecular docking analysis. Spike protein led to increased intestinal fluid accompanied by Cl- secretion, followed by intestinal edema, leukocyte infiltration, reduced glutathione levels, and increased cytokine levels (IL-6, TNF-α, IL-1β, IL-10), indicating inflammation. Additionally, the viral epitope caused disruption in the mucosal histoarchitecture with impairment in Paneth and goblet cells, including decreased lysozyme and mucin, respectively. Upregulation of TLR2 and TLR4 gene expression suggested potential activation of local innate immunity. Moreover, this experimental model exhibited reduced contractile responses in jejunal smooth muscle. In barrier function, there was a decrease in transepithelial electrical resistance and alterations in the expression of tight junction proteins in the murine jejunal epithelium. Additionally, paracellular intestinal permeability increased in human enterocytes. Finally, in silico data revealed that the Spike protein interacts with CFTR and CaCC, inferring its role in the secretory effect. Taken together, all the events observed point gut impairment, affecting the mucosal barrier to the innermost layers, establishing a successful experimental model for studying COVID-19 in the GI context.