Trophinin-associated protein (TROAP) is a cytoplasmic protein required for microtubular cytoskeleton regulation and spindle assembly, and its expression plays a critical role in the initiation and progression of various types of cancer. However, little is known about the role of TROAP in breast cancer (BC). TROAP mRNA expression levels and clinical data from Gene Expression Omnibus (GEO) datasets (GSE42568, 104 BC patients; GSE1456, 159 BC patients; and GSE21653, 266 BC patients) were analyzed by the R2: Genomics Analysis and Visualization Platform to estimate overall survival (OS). We also analyzed the genes correlated with TROAP by gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to predict potential relationships between TROAP and other genes in BC. Our study verified that both TROAP mRNA and protein expression levels were upregulated in human BC samples and cell lines. In vitro experiments demonstrated that TROAP knockdown significantly inhibited cell proliferation, the G1 to S phase transition, and the migration and invasion abilities of BC cells. The present study suggests that TROAP plays an important role in promoting the proliferation, invasion, and metastasis of BC.