Abstract Human milk (HM) contains a large variety of immune components providing protection to the infant. The presence of the cytotoxic immune proteins perforin (Per), granulysin (Grly)and granzymes (Grzm)has not yet been shown in HM cells. Per, Grnly and Grzm (A, B, H and M) were examined in cells isolated from samples of prepartum HM (PS) collected during pregnancy and HM collected longitudinally (2–5 sampling occasions) from mothers (n=31) during the first year postpartum, including 3 samples from breast infections. Gene expression was analyzed in RNA sequencing data and using qRT-PCR. Linear modeling and principle component analysis between the genes was conducted. Flow cytometry (n=5) was performed to investigate the presence of these immune proteins in HM cells. Gene expression of all immune proteins was confirmed in PS and in HM during month 1 of lactation, where variation of expression was found between women and time lactation. Strong correlations were found between GrzmA – Per (r2=0.86), GrzmA – CD45 (r2=0.76), Per – CD45 (r2=0.68) and Per – GrzmB (r2=0.67). Comparison between healthy and mastitis samples showed a higher expression of CD45 and all immune peptides in mastitis. Moreover, flow cytometry analysis showed an increase in CD45 positive cells in mastitis in comparison with healthy participants (15.1% and 4.7% respectively of total cell amount). The presence of Per, Grly and Grzm has been confirmed in HM cells. An increase in gene expression of these immune proteins has been confirmed in 3 participants suffering from breast infection. Further investigations are required to elucidate the roles of these immune peptides for the infant and/or mother