Doxorubicin (DOX) is a widely used and effective anti-cancer drug, but it is also cardiotoxic, which can lead to heart failure, and so strategies are needed to protect the heart. Fibroblast growth factor 16 (FGF-16) is preferentially expressed and released from cardiomyocytes after birth. Evidence suggests that FGF-16 decreases the risk of heart damage and limits the negative effects of heart remodeling (hypertrophy and fibrosis) after injury in vivo. Exogenous addition of FGF-16 also increased resistance to the loss of contractility in an isolated heart model of acute DOX-induced injury. Thus, how endogenous FGF-16 production and by extension function is affected by DOX treatment is of interest. The FGF-16 gene is highly conserved between human and murine species. Alignment of sequences indicates a conserved Nkx2.5 binding site in the proximal promoter region that is associated with a previously characterized TATA box. Nkx2.5 is an important factor in vertebrate heart development and congenital disease. Furthermore, Nkx2.5 RNA levels are decreased with DOX treatment. Thus, the possibility that DOX negatively affects FGF-16, perhaps through an effect on Nkx2.5 levels or binding, was investigated. Neonatal rat cardiomyocytes were treated with DOX, and FGF-16 RNA levels decreased 75% within 6 hours as assessed by qPCR. Inhibition of transcription with actinomycinD had no effect on the DOX-induced decrease in FGF-16 RNA levels. Further support for an effect of DOX on FGF-16 transcription was obtained by transfection of cardiomyocytes with a hybrid 747 bp mouse FGF-16 promoter/luciferase reporter gene that was treated with DOX; a significant decrease in luciferase activity was observed. Electrophoretic mobility shift and chromatin immunoprecipitation assays suggest reduced Nkx2.5 protein-DNA interaction with this site after DOX treatment in vitro and in situ, respectively. These data indicate that DOX decreases FGF-16 RNA expression and this correlates with a decrease in Nkx2.5 levels and association with the proximal promoter region. A direct effect of Nkx2.5 on FGF-16 promoter activity awaits further testing. Thus, a decrease or loss of FGF-16 synthesis might contribute to the DOX-induced damage and/or response to injury.