Ulva prolifera is an edible and potentially economically valuable green alga. Temperature is one of the primary factors limiting the optimal quality and yields of U. prolifera. Mutants of U. prolifera show superior production and quality characteristics. For instance, the HF-6 strain with low-temperature tolerance (LTT) can survive well at temperatures as low as 12 °C. To dissect metabolic responses and identify promising metabolite markers, we compared the metabolomes of the HF-6 and wild-type (WT) strains at 12 °C. Based on orthogonal partial least squares-discriminant analysis (OPLS-DA), volcano plot, and the receiver operating characteristic (ROC) curve analysis, six potential candidate biomarkers, glucose, galactose, panose, oxalic acid, glutamic acid (Glu), and glycine were selected. Three of these markers (glucose, galactose, and panose) were sugars, indicating that high concentrations of sugars were correlated with increased low-temperature tolerance. Oxalic acid and Glu have been commonly reported to be associated with antioxidants in plants. Further studies showed that exogenous Glu could enhance the cold tolerance capacity of U. prolifera at a concentration of 0.5 mg L−1, and it also significantly increased the relative growth rate and relative electron transfer rate (rETR) of U. prolifera relative to those of the control group. Thus, the ability of U. prolifera to tolerate low temperatures may be enhanced by regulating glycometabolism and antioxidant metabolism. To our knowledge, this is the first study of the mechanism of U. prolifera tolerance to low temperature based on comparing the metabolomics of mutant and WT strains, and it provides important insights into the mechanisms involved in the low-temperature tolerance of U. prolifera. [ABSTRACT FROM AUTHOR]