Glutathione S-transferase (GST) has been established to play an important role in regulating the responses of plants to stress, although its function and mechanisms of action in the cadmium (Cd)-tolerant Lemna minor remain unclear. In this study, we sought to identify a Cd-responsive GST gene from Lemna minor for functional analysis and mechanistic characterization. We accordingly identified a member of the GST gene family, LmGSTF3, which plays a positive role in adaptation of Lemna minor to Cd. Having successfully obtained overexpressing (OE) strains via genetic transformation, we established that these strains were characterized by elevated Cd tolerance compared with the wild-type strain, as evidenced by significant increases in growth rate, chlorophyll content, antioxidant enzyme activities, and Cd removal rate. At the transcriptome level, the OE strains were found to have a stronger regulatory ability in response to Cd, particularly with respect to photoprotection, antioxidant defense, and glycolytic metabolism, which may be key factors contributing to the Cd tolerance of Lemna minor. Our findings provide a basis for further elucidating the biochemical and molecular mechanisms underlying the Cd tolerance conferred by GST genes in Lemna minor and will potentially contribute to the utilization of Lemna minor in remediating aquatic pollution.