Triple negative breast cancer (TNBC) is the subtype with the worst prognosis. In conventional therapy, high doses of paclitaxel (PX) are used, generating several adverse effects. To avoid them, metronomic therapy (MT) arises based on the administration of lower doses with short drug-free intervals. Previously we demonstrated that a metronomic combination of the muscarinic agonist carbachol (Carb) with low doses of PX exerts an antitumor effect. Breast tumor tissue also expresses nicotinic receptors (NR), and nicotine (NIC) has been associated with resistance to conventional oncological treatment. This work aims to evaluate the signaling pathways involved in the effect of MT in the absence or presence of NIC in TNBC MDA-MB231 cells. By MTT assays we determined that NIC at a concentration like that of the smoking patients´ plasma (10-7M) increased cell viability (basal:100+/-12.7%; NIC:147.8+/-13.1%). Treatment with MT (PX10-8M+Carb10-11M) decreased cell viability (MT:67.6+/-1.8%) and the presence of NIC did not reduce the effectiveness of the treatment (MT+NIC:76.4+/-4.7%). The effect of MT was due to a mechanism dependent of PLC, PKC, Ras, MEK and NF-κB, since its selective inhibitors reduced the effect (105.1+/-9.8%; 93.5+/-10.5%; 91.8±12.3%; 90.9± 10.1% and 90.0+/-11.0% respectively). These values did not vary significantly when cells were treated in the presence of NIC. Since the mediators involved in the effect of TM are associated with the activation of nitric oxide synthase (NOS), using selective inhibitors we determined the participation of the NOS2 and 3 isoforms, which produce an increase of 114% in nitric oxide (NO) level that would be at least partially responsible for the increased levels of apoptosis. These results indicate that in TNBC MDA-MB231 cells, TM exerts its proapoptotic antitumor effect by increasing the levels of NO produced by the NOS2 and 3 isoforms in the presence or absence of NIC, highlighting the benefits of TM in these conditions