Antisense transcripts play an important role in generating regulatory non-coding RNAs but if these transcripts are also translated and for what purpose is yet poorly understood. Isolation of polysome fractions followed by RNA sequencing and mass spectrometry analysis of a six frame data-base revealed nascent peptides originating from different reading frames of bi-directional transcripts. Two peptides originating from the antisense strand stimulated the proliferation of CD8+ T cells when presented to peripheral blood mononuclear cells (PBMCs) from nine healthy donors. An antigenic peptide derived from the reverse strand of two cDNA constructs was presented on MHC-I molecules and induced CD8+ T cell activation. These results demonstrate that three frame translation of bi-directional transcripts generate antigenic peptide substrates for the immune system. This discovery holds significance for better understanding the origin of self-discriminating peptide substrates for the MHC-I pathway and for enhancing immune-based therapies against infected or transformed cells.