This experiment was conducted to investigate the effects of heat stress (HS) on the feed intake, milk production and composition and metabolic alterations in the mammary gland of dairy cows.  Twenty Holstein cows were randomly assigned to one of two treatments according to a completely randomized design.  Half of the cows were allocated to the HS group in August (summer season), and the other half were assigned to the HS-free group in November (autumn season).  HS reduced (P<0.01) dry matter intake (DMI), milk yield, milk protein and milk urea nitrogen (MUN) of cows compared with HS-free control, but increased (P<0.01) milk somatic cell counts (SCC).  We determined the HS-induced metabolic alterations and the relevant mechanisms in dairy cows using liquid chromatography mass spectrometry combined with multivariate analyses.  Thirty-four metabolites were identified as potential biomarkers for the diagnosis of HS in dairy cows.  Ten of these metabolites, glucose, lactate, pyruvate, lactose, β-hydroxybutyrate, citric acid, α-ketoglutarate, urea, creatine, and orotic acid, had high sensitivity and specificity for HS diagnoses, and seven metabolites were also identified as potential biomarkers of HS in plasma, milk, and liver.  These substances are involved in glycolysis, lactose, ketone, tricarboxylic acid (TCA), amino acid and nucleotide metabolism, indicating that HS mainly affects lactose, energy and nucleotide metabolism in the mammary gland of lactating dairy cows.  This study suggested that HS might affect milk production and composition by affecting the feed intake and substance metabolisms in the mammary gland tissue of lactating dairy cows.