The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) 1–3 enzymes confer oxygen sen-sitivity to the HIF pathway and are novel therapeutic targets for treatment of renal anemia. Inhibition of thePHDs may further be beneficial in other hypoxia-associated diseases, including ischemia and chronic in-flammation. Several pharmacologic PHD inhibitors (PHIs) are available, but our understanding of their selectivity and its chemical basis is limited.We here report that the PHI JNJ-42041935 (JNJ-1935) is structurally similar to the firefly luciferase substrate D-luciferin. Our results demonstrate that JNJ-1935 is a novel inhibitor of firefly luciferase enzymatic activity. In contrast, the PHIs FG-4592 (roxadustat) and FG-2216 (ICA,BIQ,IOX3,YM311) did not affect firefly luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of 1.36 μM. D-luciferin did not inhibit the PHDs, despite its structural similarity to JNJ-1935. This study provides insights into a previously unknown JNJ-1935 off-target effect as well as into the chemical requirements for firefly luciferase and PHD inhibitors and may inform the development of novel compounds targeting these enzymes.