Dipfluzine hydrochloride, diphenylpiperazine calcium channel blocker, is a promising candidate to treat ischemic stroke. The purpose of the study is to evaluate the acute toxicity and toxicokinetics of dipfluzine hydrochloride after single intravenous doses in rats. Acute toxicity study was performed in rats at doses of 5, 6, 10, 15, 25, 30, 35, and 40 mg/kg. Concentrations of dipfluzine in plasma and tissues were determined with a reverse-phase HPLC method after single doses of 5, 15 and 30 mg/kg. The results demonstrated that no-observed-adverse-effect level (NOAEL), lowest-observed-adverse-effect level (LOAEL), maximal tolerance dose (MTD), and minimal lethal dose (MLD) were respectively 5, 6, 30, 35 mg/kg for i.v. administration of dipfluzine hydrochloride. The toxicokinetic study revealed that the severity of toxicity was linear with the level of systemic exposure. The highest tissue exposure was detected in lung tissue and it may primarily contribute to the pulmonary congestion in dead rats. Longer elimination half-lives of dipfluzine in kidney, brain, liver, and pancreas imply a possible accumulation of dipfluzine in these tissues for long-term exposure. In addition, a temporary impairment in liver and heart was observed for clinical chemistry in 30 mg/kg dose group. The findings will help to design further studies to characterize the repeat-dose toxicity of dipfluzine hydrochloride.