The importance of the IFN-induced oligoadenylate synthetase (OAS) proteins and the OAS/RNase L pathway in the innate response against viral pathogens is well-established, however the observed differences in anti-viral activity between the human OAS1 p46 and p42 isoforms are not fully understood. The protein expression of these isoforms is determined by the SNP rs10774671, either being an A or a G allele resulting in expression of either the p42 or the p46 isoform. Using fluorescence microscopy and immunoblot analysis of fractionated cell samples, we show here that the CaaX motif is of key importance to the cellular localization. The OAS1 p42 isoform is mainly located in the cytosol, whereas the p46 isoform with a C-terminal CaaX motif is translocated to membranous organelles, like the mitochondria. We furthermore observed differences between p42 and p46 in their effect on mitochondrial physiology using high resolution respirometry and fluorometry. Overexpression of OAS1 p42 and IFN-β treatment of HeLa cells (AA genotype) resulted in significantly increased respiration, which was not seen with p46 overexpression. The difference in subcellular localization and mitochondrial effect of these two OAS1 isoforms might help to explain the anti-viral mechanisms that differentiate these proteins.