Interleukin-34 (IL-34) is a novel cytokine, which is composed of 222 amino acids and forms homodimers. It binds to the macrophage colony-stimulating factor (M-CSF) receptor and plays an important role in innate immunity and inflammatory processes. In the present study, we identified the completed IL-34 gene in 25 various mammalian genomes and found that IL-34 existed in all types of vertebrates, including fish, amphibians, birds and mammals. These species have a similar 7 exon/6 intron gene organization. The phylogenetic tree indicated that the IL-34 gene from the primate lineage, rodent lineage and teleost lineage form a species-specific cluster. It was found mammalian that IL-34 was under positive selection pressure with the identified positively selected site, 196Val. Fifty-five functionally relevant single nucleotide polymorphisms (SNPs), including 32 SNPs causing missense mutations, 3 exonic splicing enhancer SNPs and 20 SNPs causing nonsense mutations were identified from 2,141 available SNPs in the human IL-34 gene. IL-34 was expressed in various types of cancer, including blood, brain, breast, colorectal, eye, head and neck, lung, ovarian and skin cancer. A total of 5 out of 40 tests (1 blood cancer, 1 brain cancer, 1 colorectal cancer and 2 lung cancer) revealed an association between IL-34 gene expression and cancer prognosis. It was found that the association between the expression of IL-34 and cancer prognosis varied in different types of cancer, even in the same types of cancer from different databases. This suggests that the function of IL-34 in these tumors may be multidimensional. The upstream transcription factor 1 (USF1), regulatory factor X-1 (RFX1), the Sp1 transcription factor 1 , POU class 3 homeobox 2 (POU3F2) and forkhead box L1 (FOXL1) regulatory transcription factor binding sites were identified in the IL-34 gene upstream (promoter) region, which may be involved in the effects of IL-34 in tumors.