The signaling molecule 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been described as the “anti-inflammatory prostaglandin.” Here we show that substrates of the nuclear export receptor CRM1 accumulate in the nucleus in the presence of 15d-PGJ2, identifying this prostaglandin as a regulator of CRM1-dependent nuclear protein export that can be produced endogenously. Like leptomycin B (LMB), an established fungal CRM1-inhibitor, 15d-PGJ2 reacts with a conserved cysteine residue in the CRM1 sequence. This covalent modification prevents the formation of nuclear export complexes. Cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of LMB and 15d-PGJ2, demonstrating that the same single amino acid is targeted by the two compounds. Inhibition of the CRM1 pathway by endogenously produced prostaglandin and/or exogenously applied 15d-PGJ2 may contribute to its anti-inflammatory, anti-proliferative, and anti-viral effects.