Monoterpenoid indole alkaloids (MIAs) constitute one of the largest and most structurally diverse classes of alkaloids found in nature, with significant pharmacological applications. While significant discoveries have been made in MIA biosynthesis, substantial gaps remain in our understanding of biosynthetic compartmentalization and the evolution of MIA biosynthesis. In this study, we identify and characterize the yohimban O-acetyltransferase (YAT), a BAHD-type acyltransferase tightly clustered with yohimban synthase (YOS) within the reserpine biosynthetic gene cluster (BGC) of Rauvolfia. YAT specifically acetylates yohimbine and alloyohimbine in R. serpentina leaves, representing a previously unrecognized bifurcation in yohimban alkaloid metabolism. Notably, YAT exhibits a leaf-specific expression pattern, while other genes within the reserpine BGC are predominantly expressed in roots, mirroring alkaloid accumulation trends. Homology modeling and substrate docking experiments further elucidate YAT's active site, providing insights into substrate specificity and catalysis. Our findings establish the enzymatic basis of Rauvolfia MIA biosynthesis and offer insights into the evolutionary dynamics of acetyltransferases in shaping alkaloid diversity. This work also provides a foundation for synthetic biology strategies to engineer acetylated yohimban alkaloids in heterologous systems.