Water-accommodated fractions (WAFs) of crude oil include chemicals that are potent toxicants in fish. Increasing oil pollution thus demands a better understanding of molecular mechanisms for detoxification, metabolism, toxicity, and adaptation of fish. Previous studies with fish show modulation of expression of key genes in relation to stress response against WAF exposure, but there is still a lack of studies on responses of cytochrome P450 (CYP) genes and changes in biotransformation upon WAF exposure. In this study, we used the full spectrum of CYP genes of the marine medaka, Oryzias melastigma, to understand their potential mode of action on WAFs-triggered molecular mechanisms. We also analyzed further CYP-involved detoxification and endogenous steroidogenic metabolism after exposure to different concentrations of WAFs over different time courses in the marine medaka. Also, detoxification- and antioxidant-related enzymes' activities were analyzed with different concentrations of WAFs. As a result, the WAF exposure induced CYP-involved detoxification mechanism but reduced CYP-involved steroidogenic metabolism in the marine medaka. These data suggest that whole CYP profiling can be a way of understanding and uncovering the mode of action particularly with respect to emerging chemicals such as WAF exposure with the new finding that WAFs have dual functions on CYP-involved metabolisms.