Enzymatic conversion of abundant fatty acids (FAs) through fatty acid hydratases (FAHs) presents an environment-friendly and efficient route for production of high-value hydroxy fatty acids (HFAs). However, a limited diversity was achieved among HFAs to date with respect to chain length and hydroxy group position, due to high substrate- and regio-selectivity of hydratases. In this study, we compared two highly similar FAHs from Lactobacillus acidophilus: FA-HY2 has narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilize longer chain substrates and hydrate various double bond positions. We reveal three active-site residues that play remarkable role in directing substrate specificity and regioselectivity of hydration. When these residues on FA-HY2 are mutated to the corresponding residues in FA-HY1, we observe a significant expansion of substrate scope and distinct shift and enhancement in hydration of double bonds towards omega-end of FAs. A three-residue mutant of FA-HY2 (TM-FA-HY2; T391S/H393S/I378P) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting ratio of the HFA product regioisomers (10-OH:13-OH) from 99:1 to 12:88. Although kcat values are still low in comparison to wild-type FA-HY1, TM-FA-HY2 exhibited about 60-fold increase in catalytic efficiency (kcat/Km) compared to wild-type FA-HY2. Important changes in regioselectivity were also observed with mutant enzymes for arachidonic acid and C18 PUFAs. In addition, TM-FA-HY2 variant exhibited high conversion rates for cis-5, cis-8, cis-11, cis-14, cis-17-eicosapentaenoic acid (EPA) and cis-8, cis-11, cis-14-eicosatrienoic acid (ETA) at preparative scale and enabled isolation of 12-hydroxy products with moderate yields. Furthermore, we demonstrated the potential of microalgae as a source of diverse FAs for HFA production. Our study paves the way for tailor-made FAH design and for efficient conversion of FA sources into diverse range of HFAs with high potential for various applications from polymer industry to medical field.