The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant toxicant that mediates carcinogenic effects associated with oxidative DNA damage. Docosahexaenoic acid (DHA) with antioxidant functions has many biochemical, cellular, and physiological functions for cells. The present study assessed, for the first time, the ameliorative effect of DHA in alleviating the toxicity of TCDD on primary cultured rat hepatocytes (HEPs). In vitro, isolated HEPs were incubated with TCDD (5 and 10 M) in the presence and absence of DHA (5, 10, and 20 M) for 48 h. The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl) 2,5diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release. DNA damage was analyzed by liver micronucleus assay and 8-oxo-2-deoxyguanosine (8-OH-dG) level. In addition, total antioxidant capacity (TAC) and total oxidative stress (TOS) were assessed to determine the oxidative injury in HEPs. The results of MTT and LDH assays showed that TCDD decreased cell viability but not DHA. On the basis of increasing treatment concentrations, the dioxin caused significant increases of micronucleated HEPs and 8-OH-dG as compared to control culture. TCDD also led to significant increases in TOS content. On the contrary, in cultures treated with DHA, the level of TAC was significantly increased during treatment in a concentration-dependent fashion. DHA showed therapeutic potential against TCDD-mediated cell viability and DNA damages. As conclusion, this study provides the first evidence that DHA has protective effects against TCDD toxicity on primary cultured rat hepatocytes.