Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.