Diarrhea is the most prevalent side effect of afatinib as an EGFR tyrosine kinase inhibitor. The current study is to investigate the potential protective mechanisms of crofelemer or loperamide in an animal model of afatinib-induced diarrhea. Rats were randomized as the control, afatinib (50 mg/kg), afatinib plus loperamide (50 mg/kg) or afatinib plus crofelemer (50 mg/kg) group. Rats received drugs or saline through oral gavage daily for consecutive 7 days. Diarrhea, weight, serum biomarkers, gut histology or ultrastructure and multiomic changes were analyzed daily or at day 8. Afatinib induced significant diarrhea, weight loss, elevated serum levels of endotoxin, IL-6, IL-1β and TNF-α in rats. Mucosal damage was most prominent in distal ileum, showing edema, inflammatory infiltration, epithelial villus atrophy or fusion and loss of tight junction. Both loperamide and crofelemer conferred protection against afatinib-induced diarrhea and gut damage. Transcriptomic enrichment analysis showed that PPAR and IL-17 signaling pathway are among the top modified pathways in the ileum and colon of the afatinib group, respectively. Metabolomic profiling identified 318 differently abundant metabolites when comparing the afatinib and the control groups, with the most prominent enriched metabolic pathways being metabolism of xenobiotics by cytochrome P450, retinol metabolism and lysine degradation. Afatinib significantly decreased microbial diversity, which is not fully restored by administration of crofelemer or loperamide. Correlation analysis showed that cecal microbiota were significantly correlated with metabolite profiles. Loperamide and crofelemer attenuate afatinib-induced diarrhea and intestinal damage in rats, possibly through regulating microbiota-metabolic axis.