Abstract Biosynthesis of steroids by artificially designed cell factories often involves numerous nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzymes that mediate electron transfer reactions. However, the unclear mechanisms of electron transfer from regeneration to the final delivery to the NADPH-dependent active centers limit systematically engineering electron transfer to improve steroids production. Here, we elucidate the electron transfer mechanisms of NADPH-dependent enzymes for systematically engineer electron transfer of Saccharomyces cerevisiae, including step-by-step engineering the electron transfer residues of 7-Dehydrocholesterol reductase (DHCR7) and P450 sterol side chain cleaving enzyme (P450scc), electron transfer components for directing carbon flux, and NADPH regeneration pathways, for high-level production of the cholesterol (1.78 g/L) and pregnenolone (0.83 g/L). The electron transfer engineering (ETE) process makes the electron transfer chains shorter and more stable which significantly accelerates deprotonation and proton coupled electron transfer process. This study underscores the significance of ETE strategies in steroids biosynthesis and expands synthetic biology approaches.