The catalytic activity of CYP175A1 toward monooxygenation of saturated and monounsaturated fatty acids of various chain lengths (C16-C24) has been investigated to assess the enzymatic properties of this orphan thermostable cytochrome P450 enzyme. The results showed that the enzyme could catalyze the reaction of monounsaturated fatty acids but not of saturated fatty acids. The product analyses using ESI-MS and GC-MS revealed an important regioselectivity in the CYP175A1 catalyzed monooxygenation of the monoenoic fatty acids depending on the ethylenic double bond (C═C) configuration. When the double bond was in cis-configuration, an epoxy fatty acid was found to be the major product and two allyl-hydroxy fatty acids were found to be the minor products. But when the double bond was in trans-configuration the product distribution was reversed. The oxygenation efficiency was found to be the highest for palmitoleic acid (chain length C16), but there was no direct correlation of the activity with the chain length or the position of unsaturation of the fatty acid. Molecular docking calculations showed that the "U"-type conformations of the monoenoic fatty acids are particularly responsible for their binding in the enzyme pocket, and that is also consistent with the observed regioselectivity in the oxygenation reaction. The present results provide evidence that CYP175A1 can catalyze the regioselective oxygenation reaction of several monoenoic fatty acids though it cannot catalyze the oxygenation of the corresponding saturated analogues. These studies may provide critical information on the nature of the enzyme pocket and of the possible natural substrate of this orphan enzyme.