In China’s swine industry, copper is generally supplemented above the National Research Council (NRC) requirement (2012) because of its antimicrobial properties and the potential for growth promotion. Yet few are concerned about whether this excess supplementation is necessary. In this study, the 16S rRNA pyrosequencing was designed and used to investigate the effect of dietary copper level on the diversity of the fecal microbial community and the correlation of copper level with the serum level of inflammatory cytokines in Sprague-Dawley rat models. The results showed that the diet containing a high level of Cu (120 and 240 mg/kg) changed the microbial richness and diversity of rat feces associated with the increased copper content in the rat ileac and colonic digesta. Furthermore, a Pearson’s correlation analysis indicated that an accumulation of unabsorbed copper in the chyme was correlated with the microbial composition of the rat feces, which was linked with TNF-α in serum. The results suggest that dietary copper level may have a direct impact on circulating inflammatory cytokines in the serum, perhaps inducing an inflammatory response by altering the microbial composition of rat feces. Serum TNF-α could be the chief responder to excessive copper exposure.