Neoantigens are tumour-specific antigens that arise from non-synonymous mutations in tumour cells. However, their effect on immune responses in the tumour microenvironment remains unclear in breast cancer. We performed whole exome and RNA sequencing of 31 fresh breast cancer tissues and neoantigen prediction from non-synonymous single nucleotide variants (nsSNVs) among exonic mutations. Neoantigen profiles were determined by predictive HLA binding affinity (IC50 < 500 nM) and mRNA expression with a read count of ≥ 1. We evaluated the association between neoantigen load and expression levels of immune-related genes. Moreover, using primary tumour cells established from pleural fluid of a breast cancer patient with carcinomatous pleurisy, we induced cytotoxic T lymphocytes (CTLs) by coculturing neoantigen peptide-pulsed dendritic cells (DCs) with autologous peripheral lymphocytes. The functions of CTLs were examined by cytotoxicity and IFN-γ ELISpot assays. Neoantigen load ranged from 6 to 440 (mean, 95) and was positively correlated to the total number of nsSNVs. Although no associations between neoantigen load and mRNA expression of T cell markers were observed, the coculture of neoantigen-pulsed DCs and lymphocytes successfully induced CTLs ex vivo. These results suggest that neoantigen analysis may have utility in developing strategies to elicit T cell responses.