Polymorphic non-coding variants at the NOS1AP locus have been associated with the common cardiac, metabolic and neurological traits and diseases. Although, in vitro gene targeting-based cellular and biochemical studies have shed some light on NOS1AP function in cardiac and neuronal tissue, to enhance our understanding of NOS1AP function in mammalian physiology and disease, we report the generation of cre recombinase-conditional Nos1ap over-expression transgenic mice (Nos1ap (Tg)). Conditional transgenic mice were generated by the pronuclear injection method and three independent, single-site, multiple copies integration event-based founder lines were selected. For heart-restricted over-expression, Nos1ap (Tg) mice were crossed with Mlc2v-cre and Nos1ap transcript over-expression was observed in left ventricles from Nos1ap (Tg); Mlc2v-cre F1 mice. We believe that with the potential of conditional over-expression, Nos1ap (Tg) mice will be a useful resource in studying NOS1AP function in various tissues under physiological and disease states.