Steroid derivatives are polycyclic compounds with a wide spectrum of biological activities, such as anticancer, antibacterial, antioxidant, antiviral, antiparasitic, and anti-inflammatory. Several studies suggest that steroid derivatives can be effective inhibitors of aldo-keto reductases (AKRs): NADPH-dependent oxidoreductases involved in the biosynthesis and metabolism of steroid hormones, as well as in the detoxification of xenobiotics. Some AKR isoforms are overexpressed in human tumors, such as breast, prostate and colon cancer, glioma, neuroblastoma and acute myeloid leukemia. Substrates for AKRs may include carbonyl-containing cytostatic agents, leading to the inactivation of these drugs and chemoresistance. Specific inhibition of these metabolic enzymes has been established as an attractive strategy for anti-cancer drug development. In this study, the inhibition potential of D-ring modified steroids against the AKR1C4 isoform was evaluated by monitoring the decrease in NADPH fluorescence associated with AKR1C4 activity. His-tagged human recombinant AKR1C4 was expressed in Escherichia coli BL21 cells, purified using immobilized metal affinity chromatography, followed by size-exclusion chromatography. Kinetic characterization of AKR1C4 was performed by a fluorimetric assay using 9,10-phenantrenequinone (PQ) as a substrate. In the presence of several steroid derivatives, reduction of PQ was inhibited, suggesting that these steroid derivatives are promising AKR1C4 inhibitors. These preliminary results could serve as a starting point for the design of novel anticancer drug candidates targeting AKRs with high specificity. It was also shown that hepatic AKR1C4 participates in the reduction of naltrexone, an opioid receptor antagonist used in the treatment of addiction, which further indicates the importance of identifying new inhibitors of this enzyme.