Lactation activity is extremely important for dairy cows, but the underlying metabolic mechanisms is not well understood. This study was conducted to investigate the lactation-related maintenance and initiation metabolic mechanisms in dairy cow using overall biofluid and partial tissue metabolomics. Six mid-lactation Holstein cows were used to analyze the relationships between 4 biofluids (rumen fluid, serum, milk and urine) and to compare mammary gland metabolome with 6 nonlactating cows using gas chromatography-time of flight/mass spectrometry and multivariate analysis. Totally, 33 mutual metabolites and 274 metabolites were identified in 4 biofluids and mammary gland tissues, respectively. The sub-clusters of heatmap analysis for rumen fluid and serum were grouped together and highly correlated with each other, but separated from milk. Creatine was identified as key metabolite to explain the biological variation among 4 biofluids. Pathways of gluconeogenesis, pyruvate metabolism, tricarboxylic acid (TCA) cycle, glycerolipid metabolism and aspartate metabolism, demonstrated most functional enrichment among 4 biofluids (false discovery rate <0.05, fold enrichment >2). Clear discriminations were observed between lactating and nonlactating cows, with 54 significantly higher (P < 0.05, VIP >1) metabolites in lactation group. Lactobionic acid, citric acid, orotic acid and oxamide were extracted by S-plot as putative biomarkers. The TCA cycle, glyoxylate and dicarboxylate metabolism, glutamate metabolism and glycine metabolism were determined as functional impact pathways (P < 0.01, impact value >0.1) in lactation group. Extremely upregulated function of the TCA cycle pathway (P < 0.0001) in lactating cows was identified along with 70% substrates increased in the mammary gland cell. These results provide the first integrated insight into better understanding of lactation-related overall and partial metabolic mechanisms and will be beneficial in developing regulated strategies for lactating dairy cows. More importantly, novel systematic investigation can be obtained from this study to address complex biological questions.