Enzymes are generally believed to be highly regio- and stereoselective catalysts that strictly control the reaction coordinates and dominate the final catalytic outcomes. However, recent studies have started to suggest that substrates sometimes play key roles in determining the product selectivity in enzyme catalysis. Here, we highlight several enzymatic reactions in which the stereoselectivity is, at least in large part, governed by the intrinsic properties of the substrate rather than by characteristics of the enzyme. These reactions are involved in the biosynthesis of different classes of natural products, including lanthipeptides, sactipeptides, and polyketides. Understanding the mechanism of substrate-controlled stereospecificity may not only expand our knowledge of enzyme catalysis and enzyme evolution but also guide bioengineering efforts to produce novel valuable products.