Cucurbitacins are highly oxygenated triterpenoids characteristic of plants in the family Cucurbitaceae and responsible for the bitter taste of these plants. Fruits of bitter melon (Momordica charantia) contain various cucurbitacins possessing an unusual ether bridge between C5 and C19, not observed in other Cucurbitaceae members. Using a combination of next-generation sequencing and RNA-Seq analysis and gene-to-gene co-expression analysis with the ConfeitoGUIplus software, we identified three P450 genes, CYP81AQ19, CYP88L7, and CYP88L8, expected to be involved in cucurbitacin biosynthesis. CYP81AQ19 co-expression with cucurbitadienol synthase in yeast resulted in the production of cucurbita-5,24-diene-3β,23α-diol. A mild acid treatment of this compound resulted in an isomerization of the C23-OH group to C25-OH with the concomitant migration of a double bond, suggesting that a nonenzymatic transformation may account for the observed C25-OH in the majority of cucurbitacins found in plants. The functional expression of CYP88L7 resulted in the production of hydroxylated C19 as well as C5-C19 ether-bridged products. A plausible mechanism for the formation of the C5-C19 ether bridge involves C7 and C19 hydroxylations, indicating a multifunctional nature of this P450. On the other hand, functional CYP88L8 expression gave a single product, a triterpene diol, indicating a monofunctional P450 catalyzing the C7 hydroxylation. Our findings of the roles of several plant P450s in cucurbitacin biosynthesis reveal that an allylic hydroxylation is a key enzymatic transformation that triggers subsequent processes to produce structurally diverse products.