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Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.