To clarify the impact of dicoumarin (DIC) on the ovarian cells metabolome of Spodoptera litura (SL-221), this study employed CCK-8 and non-targeted metabolomics techniques to investigate the toxicological mechanism of DIC in inhibiting SL-221 cells proliferation. The results showed that DIC exhibited obvious inhibitory activity against SL-221 cells, with an inhibitory concentration of 1.60 µg/mL at 24 h, and the inhibition was concentration-dependent. Furthermore, when co-cultured with SC 79 and insulin respectively, DIC enhanced cell viability. Through non-targeted metabolomics analysis, a total of 393 differential metabolites were obtained, with 364 DIC upregulated and 29 DIC down-regulated compared to those of the control group. More than 50% differential metabolites were distributed in amino acids and their metabolites (48.3%) and glycerophospholipids (14.2%). The differential metabolites were mainly enriched in nutritional metabolic pathway of amino sugar and nucleotide sugar metabolism, unsaturated fatty acid biosynthesis, sugar biosynthesis, starch and sucrose. Among them, the differential metabolites adrenosterone, LPC (12:0/0:0), N, N-dicyclohexylcarbodiimide, and Val-Pro upregulated by 5 833.56, 15.47, 10.26, and 9.94 times, respectively. This study elucidated the metabolic regulatory mechanism of DIC-induced SL-221 cell proliferation and provided a theoretical basis for the development and utilization of this compound.