Calf diarrhea caused by pathogenic Escherichia coli is a major cause of death in calves, with a mortality rate of over 50%. It is crucial to understand the pathogenesis and development of calf diarrhea for its prevention and treatment. We aimed to study the effect of pathogenic E. coli on the flora composition, function, and short-chain fatty acid (SCFA) content of calf feces using a calf diarrhea model. Sixty-four newborn Holstein calves (40–43 kg) were divided into a normal group (NG; n = 32) and a test group (TG; n = 32). At the beginning of the experiment, the TG were orally administered pathogenic E. coli O1 (2.5 × 1011 CFU/mL, 100 mL) to establish a calf diarrhea model, and the NG were orally administered the same amount of physiological saline solution. The calves of the two groups were subjected to the same feeding and management. Fresh feces samples were collected at different time points and subjected to 16S rRNA high-throughput sequencing and gas chromatography–mass spectrometry to determine the fecal microbial composition and SCFA content. Pathogenic E. coli O1 significantly altered microbiotas composition in the feces of calves, increasing the relative abundance of Proteobacteria and decreasing that of Firmicutes. It also led to a significant increase in the relative abundance of Escherichia-Shigella and a decrease in Lactobacillus, as well as significantly decreased SCFA content. Therefore, we postulate that pathogenic E. coli induces calf diarrhea by causing intestinal florae imbalance and reducing the content of SCFA.