This study was to clarify whether acrylamide (ACR) will induce apoptosis and astrogliosis in an astrocytic cell line in vitro. Different time- and dose-dependent cytotoxic studies were conducted upon neuronal (SH-SY5Y) and glial cell lines (U-1240 MG) under exposure to ACR up to 72 h. We showed that SH-SY5Y cells were more sensitive in cytotoxic assays than U-1240 MG cells, and significantly decreased cell viability was observed at concentrations higher than 1 mM with increased lactate dehydrogenase leakage observed only at 5 and 10 mM in U-1240 MG cells. The ACR-induced apoptotic responses and phosphorylation of p53 protein at Ser15 for U-1240 MG cells were identified at 48 h. The increase of glial fibrillary acidic protein (GFAP) as a chemical-induced astrogliotic response was found to be associated with different ACR concentrations and exposure times, particularly at ≧48 h of ≧2 mM. In addition, immunocytochemical staining at 36 h of 5 and 10 mM treatments had significantly higher density of GFAP than the control. Thus, ACR-induced effects can be seen in neuronal and astrocytic cells. These results suggest that ACR exposure may lead to apoptotic and astrogliotic effects in human astrocytoma cells in vitro in a time- and dose-dependent manner.