Glioma patients constitute the greatest percentage of depressed neoplasm patients. These patients often require antidepressant treatment, but the effect of antidepressant drugs on glioma cells requires further evaluation. In the present study, we evaluated the effect of trifluoperazine (TFP) on the proliferation and apoptosis of glioma cells. Transcriptomic and bioinformatics analysis results suggested that antidepressant drugs, especially TFP, may upregulate the drug-resistant ability of glioma cells. A low concentration of TFP upregulated the viability of glioma cells. Colony formation and EdU assays confirmed that TFP treatment accelerates glioma cell proliferation, but no significant difference was found in the cell cycle distribution of glioma cells after treatment with TFP or control. Flow cytometry and TUNEL staining results suggested that TFP treatment decreased apoptosis in glioma cells. In addition, TFP treatment downregulated the intracellular Ca²⁺ concentration of glioma cells. In vivo experimental results indicated that TFP treatment promoted proliferation and reduced apoptosis in xenograft tumours in nude mice. Taken together, our results suggest that a low concentration of TFP promotes proliferation and reduces apoptosis in glioma cells both in vitro and in vivo. The potential harmful effects of antidepressant drugs on gliomas require further evaluation before their use in glioma patients.