(1) Background: The functions of the tropomyosin (TPM) family in tumors and cancers have been explored; however, striking differences have been observed. This study aims to further our understanding of the effects of TPMs in glioma, and find novel biomarkers for glioma. (2) Methods: RNA-seq data were downloaded from TCGA and GTEx. Survival analyses, Cox regression, nomogram, calibration curves, ROC curves, gene function enrichment analyses, and immune cell infiltration analyses were carried out using R. CCK8 assay, while Brdu assay, colony formation assay, and Transwell assay were used to verify the functions of TPM3 in glioma. (3) Results: TPM1/3/4 were significantly more highly expressed in glioma than that in normal tissues, while higher expression of TPM2/3/4 was correlated with a worse overall survival than lower expression of TPM2/3/4. Furthermore, bioinformatic analyses indicated that TPM3/4 could be promoting factors for poorer survival in glioma, but only TPM3 could serve as an independent prognostic factor. Gene function analyses showed that TPMs may be involved in immune responses. Moreover, further experimental investigations verified that TPM3 overexpression enhanced the proliferation and tumorigenicity of glioma. (4) Conclusions: High expression of TPM3/4 was positively correlated with poorer prognosis in glioma, and TPM3 could serve as a novel independent prognostic factor of glioma.