Transport stress not only causes physiological changes but also induces behavioral responses, including anxiety-like and depression-like behavioral responses in animals. The neuronal nitric oxide synthase (nNOS) plays a pivotal role in transport stress. This study aimed to investigate the effects of acute transport stress on the expression of nNOS and the distribution of nNOS-positive neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus in rats and to explore the neuroendocrine mechanism of transport stress.In this study, for the first time, we investigated the effects of transport stress on nitric oxide (NO)-NOS in the hypothalamus. After simulated stress, rats exhibited behavioral changes in the open field test (OFT), increased serum corticosterone (CORT) and norepinephrine (NE) levels, and increased NO content in the hypothalamus. In addition, nNOS expression in the hypothalamic PVN was upregulated, and its distribution was altered in stressed rats compared with that of unstressed rats.Our findings indicate that simulated transport stress increases nNOS expression and alters its distribution in the PVN of the rat hypothalamus.