Linoleic acid (LA) shows great potential in inhibiting the growth of multiple red tide microalgae by disturbing algal physio-biochemical processes. However, our knowledge on the mechanisms of algal mortality at metabolic level remains limited. Herein, the response of K. mikimotoi to LA was evaluated using metabolomics, stable isotope techniques (SIT), and physiological indicators. Results showed that 100 μg/L LA promoted the growth of K. mikimotoi, which was significantly inhibited by 500 μg/L LA, along with a significant reduction of photosynthetic pigments and a significant increase of reactive oxygen species (ROS). SIT showed that LA entered algal cells, and 56 isotopologues involved in ferroptosis, carotenoid biosynthesis, and porphyrin metabolism were identified. Non-targeted metabolomics identified 90 and 111 differential metabolites (DEMs) belonging to 11 metabolic pathways under the 500 μg/L and 100 μg/L LA exposure, respectively. Among them, 34 DEMs were detected by SIT. Metabolic pathway analysis showed that 500 μg/L LA significantly promoted ferroptosis, and significantly inhibited carotenoid biosynthesis, porphyrin metabolism, sphingolipid metabolism, and lipopolysaccharide biosynthesis, presenting changes opposite to those observed in 100 μg/L LA-treated K. mikimotoi. Overall, this study revealed the metabolic response of K. mikimotoi to LA, enriching our understanding on the allelochemical mechanism of LA on K. mikimotoi.