Adrenocortical Carcinoma is a rare and aggressive endocrine malignancy, that arises from cells of one of the three cortical layers of the adrenal gland. Radical surgery is the only curative treatment, even if recurrence rates are high. Therapeutic options are limited, with mitotane as the cornerstone of medical therapy. Despite 50 years of clinical use, the mechanism of action of mitotane has not yet been fully established, possibly due to the drug’s susceptibility to interaction with confounding factors that reduce its biological activity. In the present study, we evaluated by RNAseq the effect of mitotane on gene expression in the H295R cell line, in an environment free of known confounding factors. Our approach allowed us to identify transcriptional deregulation of the ATF4/ATF3 axis, often involved in ER stress. These results were also validated by ddPCR in independent experiments. Mitotane-mediated ATF4 overexpression was also confirmed at the protein level. We observed how an incremental concentration of mitotane could deregulate main biological pathways. Further, we confirmed, both at RNAseq and ddPCR level, the mitotane-mediated downmodulation of genes such as STAR, CYP11A1, CYP21A2, and HSD3B2, highlighting its effect on steroid hormones biosynthesis. Through our approach, we identified biological pathways altered by mitotane in early response stages and with low drug concentrations. Some of these pathways could potentially be investigated in the future as functional biomarkers to monitor adrenocortical carcinoma treatment or as new pharmacological targets for this rare disease.