Gut homeostasis is of significant importance because gut microbiota affect a number of mammalian processes. However, there have been few studies on in-depth metabolomics study of homeostasis and metabolic status of gut micobiota. Due to the high sensitivity and selectivity, LC-MS based metabolomics have gone mainstream in metabolomics but still face many technical challenges, both qualitatively and quantitatively. In this study, we employed a novel staggered gas-phase fractionation (sGPF) for improved data-dependent acquisition for untargeted metabolite identification and more than 250 urinary metabolites were characterized at MS/MS level in positive ion mode. The untargeted profiles were then transformed into a targeted metabolomic approach featured with tailored sensitivity reduction which showed improved pattern recognition and information recovery. Combing the above strategies, we performed the first comprehensive investigation of urinary host-microbial co-metabolites of healthy individuals. Principal components analysis (PCA) was employed to provide both global metabolic phenotype pattern index and the host-microbial co-metabolism pattern index. Correlation analysis showed prevalent positive and negative correlations (-0.34 < R < 0.36, p < 0.05, N = 69) between the principal components of global phenotype pattern and co-metabolism pattern, indicating host-microbial cometabolism was widely associated with human phenotypes. No obvious gender- or age-related co-metabolism pattern was observed. High intra-individual and interindividual variations of host-microbial co-metabolites were also observed. Secondary metabolomics of gut microbiota isolated from fecal samples revealed distinct patterns of bacterial secondary metabolites and medium nutrients between iv patients with ulcerative colitis (UC) and healthy controls, indicating microbiota could modulate gut environment in two ways. We found gut bacteria from UC patients was deficient in the decomposition of large nutritional peptides and produced less polyunsaturated fatty acid (PUFA) derivatives. We also investigated the discrepancies and similarities between gut bacterial secondary metabolomics of human and dextran sulfate sodium (DSS) induced UC rat. The results indicated that DSS-induced UC rat evoke a less obvious microbial shift versus UC patients at secondary metabolomics level, for which the species differences (human vs rat) were more pronounced than condition differences (healthy vs UC).