Macrocyclic diterpenoid esters from Euphorbiaceae plants hold significant medicinal value owing to their structural diversity and for attributing structural uniqueness and biological efficacy. However, the responsible enzymes for the acylation of macrocyclic diterpenoids remain unknown. We identified two macrocyclic diterpenoid O-acyltransferases, ElBAHD16 and ElBAHD35, from the diterpene biosynthetic gene cluster of Euphorbia lathyris. ElBAHD16 and ElBAHD35 were characterized both in vitro (using Escherichia coli) and in vivo (using Nicotiana benthamiana and E. lathyris) and exhibited mono-acylation activities toward the hydroxy groups of their substrates, 7-hydroxylathyrol and lathyrol. ElBAHD16 showed not only regioselectivity toward the 7-OH group of 7-hydroxylathyrol but also donor promiscuity, thereby producing three different mono-acylation products. Conversely, ElBAHD35 demonstrated specific recognition for the 5-OH group of 7-hydroxylathyrol and lathyrol, thereby mediating mono-acetylation reactions with acetyl-CoA, showing donor specificity. Site-directed mutagenesis revealed that residues H154 and T363 in ElBAHD16 are critical for its catalytic activity. Notably, the Q35 residue enhanced the efficiency of ElBAHD16, while the M296, N292, and F394 residues were crucial for its donor promiscuity. These findings elucidate the last step in the biosynthesis of macrocyclic diterpenoid esters and highlight the contribution of acyltransferases to the structural diversity of diterpenoids.