Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is responsible for NTSR-1 localization and signaling within SMDs upon NTS stimulation. Site-directed mutagenesis and pharmacological strategies were utilized to assess NTRS-1 post-translational modifications in an over-expression cell model (HEK293T) as well as a native breast cancer cell model (MDA-MB-231). NTSR-1 palmitoylation was confirmed by multiple chemical and fluororadiographic methodologies. NTSR-1 glycosylation was confirmed by pharmacological (tunicamycin) and chemical (PGNaseF and O-type glycosidase) approaches. Physiological correlates including cell viability (MTS assay), apoptosis (caspase 3/7 assay) and ERK phosphorylation were utilized to assess the consequences of NTRS-1 palmitoylation. The interaction between palmitoylated NTRS-1 and Gαq/11 within SMDS was confirmed with immunopreciptation analysis of detergent-free isolated fractions of caveolin-rich microdomains. We identified dual-palmitoylation at Cys381 and Cys383 of endogenously-expressed NTSR-1 in MDA-MB-231 breast adeno-carcinomas as well as exogenously-expressed NTSR-1 in HEK293T cells (which do not normally express NTSR-1). Pharmacological inhibition of NTSR-1 palmitoylation in MDA-MB-231 cells as well as NTSR-1-expressing HEK293T cells diminished NTS-mediated ERK 1/2 phosphorylation. Additionally, NTSR-1 mutated at Cys381 and Cys383 showed diminished ERK1/2 stimulation and reduced ability to protect HEK293T cells against apoptosis induced by serum starvation. Mechanistically, mutated C381,383S-NTSR-1 showed reduced ability to interact with Gαq/11 and diminished localization to structured membrane microdomains (SMDs), where Gαq/11 preferentially resides. We also demonstrated that only glycosylated isoforms of NTRS-1 localize within SMDs by palmitotylation. Collectively, our data establish palmitoylation as a novel pharmacological target to inhibit NTSR-1 mitogenic signaling in breast cancer cells.