Purpose: As a malignancy that develops from breast tissue, breast cancer has been widely regarded as the most common type of cancer threatening the health of women worldwide. Emerging evidence has demonstrated that tsRNAs might play a vital part in the tumorigenesis and progression of several types of cancers. However, the functions of tsRNAs in breast cancer remain largely unknown. Here, we investigated the functions of tsRNA-26576 in tumorigenesis of breast cancer. Patients and methods: In this study, the tsRNA deregulation states in breast cancer patients (four cancer tissues and four adjacent normal tissues) were evaluated using small RNA sequencing. And then, RT-PCR was used to detected the tsRNA-26576 expression level in breast cancer patients. Results: A total of 263 tsRNAs were identified as significantly differentially expressed, of which 75 were upregulated, and 188 were downregulated. The functional classification through KEGG pathway database illustrated that the most significant pathway enriched by the targets of differentially expressed tsRNAs was the pathway in cancer. Among these differently expressed tsRNAs, we found that tsRNA-26576 was remarkably upregulated in cancer tissue in comparison with adjacent normal tissue. Meanwhile, RT-PCR results verified that tsRNA-26576 expression level was highly upregulated in 10 paired samples from breast cancer patients. Besides, tsRNA-26576 was found to motivate cellular multiplication and migration while suppressing cellular apoptosis in MDA-MB-231 cells. Moreover, mRNA sequencing results showed that several tumor suppressor genes, including FAT4 and SPEN, were upregulated after delivering tsRNA-26576 inhibitor in MDA-MB-231 cells. Conclusion: We found tsRNA-26576 was upregulated in breast cancer tissue, and it could promote the cell growth while inhibite cell apoptosis. Therefore, tsRNA-26576 might serve as a potential clinical therapy target and a predictive marker for breast cancer.