Cytochrome P45051(CYP51) catalyzes C-C bond cleavage during the biosynthesis of the sterol, using a mechanism which is still under debate. Moreover, the mechanistic conundrum is aggravated by the experimental results, which show that the C-C bond cleavage reaction step may start either from the gem-diol or aldehyde intermediates. The present study uses hybrid QM/MM calculations (exploration of potential energy surface) supplemented by long-scale MD simulations to elucidate the mechanism of C-C bond cleavage in 14α-demethylation of lanosterol using both gem-diol and aldehyde as starting substrates for the reaction. For gem-diol, the reaction proceeds via a proton-coupled electron transfer (PCET) type pathway. However, in the aldehyde form, demethylation follows a surprising mechanism assisted by a water molecule. In both mechanisms, the active catalyst is the classical Compound I species, [(Por+•)(Scys)FeIVO]. Since the C-C cleavage plays a crucial role in the natural biosynthesis of many complex organic molecules in the biological kingdom, the present study provides a testable mechanism of this enigmatic reaction. © 2022 American Chemical Society. All rights reserved.