Active glutamine utilization is critical for tumor cell proliferation. Glutaminolysis represents the first and rate-limiting step of glutamine utilization and is catalyzed by glutaminase (GLS). Activation of ErbB2 is one of the major causes of breast cancers, the second most common cause of death for women in many countries. However, it remains unclear whether ErbB2 signaling affects glutaminase expression in breast cancer cells. In this study, we show that MCF10A-NeuT cell line has higher GLS1 expression at both mRNA and protein levels than its parental line MCF10A, and knockdown of ErbB2 decreases GLS1 expression in MCF10A-NeuT cells. We further show that in these cells, ErbB2-mediated upregulation of GLS1 is not correlated to c-Myc expression. Moreover, activation of neither PI3K-Akt nor MAPK pathway is sufficient to upregulate GLS1 expression. Interestingly, inhibition of NF-κB blocks ErbB2-stimulated GLS1 expression, whereas stimulation of NF-κB is sufficient to enhance GLS1 levels in MCF10A cells, suggesting a PI3K-Akt-independent activation of NF-κB upregulates GLS1 in ErbB2-positive breast cancer cells. Finally, knockdown or inhibition of GLS1 significantly decreased the proliferation of breast cancer cells with high GLS1 levels. Taken together, our data indicate that ErbB2 activation promotes GLS1 expression via a PI3K-Akt-independent NF-κB pathway in breast cancer cells, identifying another oncogenic signaling pathway which stimulates GLS1 expression, and thus promoting glutamine utilization in cancer cells. These findings, if validated by in vivo model, may facilitate the identification of novel biochemical targets for cancer prevention and therapy. J. Cell. Biochem. 115: 498–509, 2014. © 2013 Wiley Periodicals, Inc.