Methanol represents a promising sustainable one-carbon (C1) feedstock for biomanufacturing. However, limitations arising from cytotoxicity and incomplete metabolic understanding have resulted in low production efficiencies and constraining industrial applications. Recent systems biology approaches, including multi-omics and computational modeling, have significantly advanced our knowledge of microbial methanol metabolism. Here, we first summarize recent methodological progress in systems biology applied to methanol metabolism studies. Secondly, we highlight novel insights into understanding methanol metabolism by applying systems biology approaches in natural and synthetic methylotrophic microorganisms. Finally, we discuss the challenges and future perspectives for systems biology studies of methanol metabolism in yeast.