Background/Objectives: Although copper nanoparticles (Cu-NPs) are increasingly explored as food and feed additives, there is still limited evidence on how the commonly consumed dietary fibre matrix modulates their effects on the gut microbiota. This study evaluated whether different dietary fibres (cellulose, pectin, inulin, psyllium) modulate Cu-NP–driven changes in caecal microbiota activity, composition, and bile acid metabolism in rats in a multifactorial design accounting for fibre type, copper dose, and copper form. Methods: Wistar male rats (n = 10 per group, 10 groups) were fed semi-purified diets for 6 weeks. Cu-NPs were provided at 6.5 or 13 mg Cu/kg diet and combined with cellulose (control fibre) or with pectin, inulin, or psyllium. Caecal digesta parameters, microbial enzyme activities, short-chain fatty acids (SCFAs), bile acids, and 16S rRNA sequencing were used to assess microbial diversity. Results: Final body weight did not differ among groups, whereas feed intake decreased most consistently with inulin and psyllium. Inulin and psyllium increased caecal digesta and tissue mass, while pectin increased caecal ammonia. Higher Cu-NPs dose reduced several microbial enzyme activities and lowered major SCFAs across most treatments; pectin most strongly preserved/enhanced glycosidase activities and was associated with increased SCFA levels vs. control, with a 32% rise in acetate, a 47% rise in propionate, and a 61% rise in butyrate. Fibre type dominated bile acid outcomes: psyllium reduced total bile acids by 11.8% vs. control, while inulin increased muricholic acids by 216% vs. control. Microbiota alpha and beta diversity separated primarily by fibre type, with distinct clustering particularly in pectin-fed groups. Across comparisons, Mucispirillum was consistently reduced in fibre-supplemented groups vs. cellulose, alongside recurrent changes in selected genera; functional profiling highlighted shared shifts in carbohydrate, fermentation, transport, and stress-response features under Cu-NPs exposure. Conclusions: The gastrointestinal and microbiota responses to Cu-NPs are strongly fibre-dependent; thus, Cu-NP safety and functionality should be evaluated together with the accompanying dietary fibre matrix, not as a standalone exposure. Implications for humans remain indirect and require confirmation in human-relevant models and clinical settings.
