Apoptosis Related Protein 3 (APR3) is an important protein which is involved in retinoic acid-induced apoptosis, osteoblast differentiation and cervical squamous cell carcinoma progression. Although it was predicted to be a trans-membrane protein, its cellular localization is not clear. In this study, we analyzed APR3 with bioinformatic tools and found that APR3 contains a potential signal peptide, a transmembrane region and 3 N-glycosylation sites, all of which are characteristics of lysosomal proteins. Western blot with isolated lysosomes demonstrated that APR3 was mainly present in lysosomes, specially in the lysosomal membrane fraction, but not in endoplasmic reticulum. Concomitantly, double immunofluorescence confirmed that APR3 co-localized with lysosomal membrane protein, LAMP1, as well as lysosomal specific marker, Lyso-Tracker Red. Moreover, we showed that APR3 was highly expressed in the lung, liver, spleen, kidney and adipose tissue, but expressed at the low level in the heart, pancreas, stomach and intestine. Interestingly, APR3 expression was elevated in multiple hepatocellular carcinoma cell lines comparing to normal liver cells. Collectively, our results proved that APR3 is a novel lysosomal membrane protein and shed light on its possible functions.