genome and has restricted the feasibility of using pigs as a biomedical model. We developed the tissue-based proteome map using 34 major normal pig tissues. A total of 5841 unknown protein isoforms were identified and systematically characterized, including 2225 novel protein isoforms, 669 protein isoforms from 460 genes symbolized beginning with LOC, and 2947 protein isoforms without clear NCBI annotation in current pig reference genome. These newly identified protein isoforms were functionally annotated through profiling the pig transcriptome with high-throughput RNA sequencing (RNA-seq) of the same pig tissues, further improving the genome annotation of corresponding protein coding genes. Combining the well- annotated genes that having parallel expression pattern and subcellular witness, we predicted the tissue related subcellular components and potential function for these unknown proteins. Finally, we mined 3081 orthologous genes for 52.75% of unknown protein isoforms across multiple species, referring to 68 KEGG pathways and 23 disease signaling pathways. These findings provided valuable insights and a rich resource for enhancing studies of pig genomics and biology as well as biomedical model application to human medicine.