Secreted proteins play important roles in many crucial biological processes, and can be new agents or targets for drug therapies. Here, we report on the isolation and characterization of a novel human non-classical secreted protein which is encoded by the hypothetical gene C19orf24 (chromosome 19 open reading frame 24). It has no signal peptide, but can still secrete extracellularly despite the presence of the inhibitor brefeldin A (BFA), proving its non-classical secreted protein status. Via subcellular localization using C19orf24 in vivo and transfected pEYFP-Golgi plasmid in Hela cells, C19orf24 was shown not to co-localize in the Golgi apparatus, which suggested that it secretes via a new and unknown pathway. Deglycosylation analysis with PNGase F verified that it has no N-glycosylation modification sites. Via the reverse transcription-PCR method, it was found to be expressed only in the human liver, and preferentially in normal tissue. In addition, C19orf24 was shown to be a recently evolved gene, found only in Homo sapiens and Pan troglodytes. By calculating its synonymous and non-synonymous substitution rate (d (S)/d (N)), we found that it experienced a purifying selection, which suggests that C19orf24 may have a special, irreplaceable biological function in the human organism.