The endoplasmic reticulum (ER) is required for the de novo biogenesis of peroxisomes in mammalian cells. However, its role in peroxisome maintenance is unclear. In order to explore ER involvement in the maintenance of peroxisomes, we redirect a peroxisomal membrane protein (PMP), PEX3, to directly target to the ER using the N-terminal ER signal sequence from preprolactin. Using biochemical techniques and fluorescent imaging, we find that the ER-targeted ssPEX3 is continuously imported into pre-existing peroxisomes. This suggests that the ER constitutively provides membrane proteins and associated lipids to pre-existing peroxisomes. Using quantitative time-lapse live-cell fluorescence microscopy applied to cells either depleted of or exogenously expressing PEX16, we find that PEX16 mediates the peroxisome trafficking of two distinct peroxisomal membrane proteins, PEX3 and PMP34, via the ER. These results not only provide insight into peroxisome maintenance and PMP trafficking in mammalian cells but also highlight important similarities and differences in the mechanisms of PMP import between the mammalian and yeast systems.