Background/Aim: Previous reports identified a global proteomic signature of estrogen-independent letrozole resistant breast cancer cells, however, it remains unclear how letrozole-resistance is impacted when cells remain estrogen receptor positive (ER+). Materials and Methods: To capture the protein expression profile associated with ER+ Aromatase inhibitor (AI) resistance, a global proteomic analysis was conducted using the letrozole-sensitive (T47Darom cells) and letrozole-resistant cells (T47DaromLR cells). To examine the molecular features associated with this phenotype Kaplan-Meier analysis, phospho-antibody arrays, proliferation and apoptosis assays were conducted. Results: MAP3K6 was upregulated in the T47DaromLR cells by 3.2-fold (p<0.01) which was associated with a decrease in relapse-free survival among breast cancer patients (p=0.0019). Members of the MAPK/p38 pathway (i.e., and p70S6K MAPK) were also increased in the T47DaromLR cells, while inhibiting p38 led to decreased proliferation and induction of apoptosis. Conclusion: Activation of the p38/MAPK pathway leads to ER+ AI-resistance.Aromatase inhibitors, like letrozole, are the first-line therapy for postmenopausal women with estrogen-dependent breast cancer. These agents block the biosynthesis of estrogen through aromatase inhibition and inhibit the proliferation of estrogen-dependent breast tumors. Unfortunately, after several years some patients develop therapeutic resistance, and the tumors continue to proliferate. To study aromatase inhibitor resistance in the laboratory setting, a letrozole resistant cell line was developed by inoculating the MCF-7Ca cells (MCF-7 cells stably transfected into the human aromatase gene) into immunocompromised mice and treating the mice with letrozole for 56 weeks (1). After long-term treatment, the tumors acquired resistance to letrozole, were cultured in vitro and termed long term letrozole treated (LTLT)-Ca cells. Previous studies in our lab have demonstrated that as the letrozole-sensitive cells (AC-1) transition to the letrozoleresistant phenotype (LTLT-Ca) they are associated with estrogen independence, up-regulation of growth factor signaling cascades, and increased motility (2). However, the changes that occur as cells acquire resistance to letrozole while maintaining estrogen dependence remains unclear.To this end, an in vitro model of letrozole resistance was developed whereby the T47D parental cell line was stably transfected with the human aromatase gene and termed the T47Darom cells. The T47Darom cells were treated with letrozole for 75 weeks until they became resistant to letrozole and were called T47DaromLR cells. When the T47DaromLR cells were characterized, they retained both aromatase activity and ER expression (3). As such, this was an ideal model to test the hypothesis that there are distinct proteomic profiles between estrogen-dependent and estrogen-independent letrozole resistant breast cancer cells, 583 This article is freely accessible online.