To date, there have been no exposure standards for air concentrations of 1,4-dichlorohexafluorobutene (DCHF) in the work areas. The study was aimed to assess the toxicity of DCHF and to evaluate health hazard in acute, subacute, and chronic experiments. It was found that the substance was highly hazardous, DL₅₀ in mice after intragastric injection was 79.0 mg/kg, СL₅₀ was 229.0 mg/m³, and in rats these values were 86,0 mg/kg and 670,0 mg/m³. In animals, DCHF had a moderate local irritative effect on animal skin and ocular mucous membranes, as well as the skin resorptive effect. The 18.2 mg/m³ threshold limit concentration for a single inhalation exposure to DCHF was defined based on the changes in behavior responses and blood parameters. The 30-day subacute inhalation experiment revealed the pronounced cumulative effect of the substance. The 4-months chronic inhalation study showed that the exposure of experimental rats to 16.8 mg/m³ concentration of DCHF resulted in impaired function of central nervous system and cardiac activity, altered hematologic, biochemical, acid-base, and blood gas values, as well as in morphological alterations in lungs, which persisted after the 30-day recovery period. The chronic exposure threshold defined for DCHF was 2.2 mg/m³, and the defined no observable effect level was 0.24 mg/m³. Based on the study results, the maximum permissible concentration of DCHF in the air of the working area of 0.2 mg/m³ was confirmed and approved, the substance was assigned hazard class 2, vapor + aerosol + (specific protection of skin and eyes required). Gas chromatographic method using electron-capture detection for determination of DCHF mass air concentration in the work areas has been developed and approved.