The aims of this work were to evaluate the expression of histamine H₃ receptor (H₃R) in triple negative breast cancer (TNBC) samples and to investigate the antitumoral efficacy and safety of the LINS01 series of H₃R antagonists, through in silico, in vitro, and in vivo approaches. Antitumor activity of LINS01009, LINS01010, LINS01022, LINS01023 was assayed in vitro in 4T1 and MDA-MB-231 TNBC cells (0.01–100 μM), and in vivo in 4T1 tumors orthotopically established in BALB/c mice (1 or 20 mg/kg). Additionally, H₃R expression was assessed in 50 human TNBC samples. We have described a higher H₃R mRNA expression in basal-like/TNBC tumors vs. matched normal tissue using TCGA Pan-Cancer Atlas data, and a higher H₃R expression in human tumor samples vs. peritumoral tissue evidenced by immunohistochemistry associated with poorer survival. Furthermore, while all the essayed compounds showed antitumoral properties, LINS01022 and LINS01023 exhibited the most potent antiproliferative effects by: i) inducing cell apoptosis and suppressing cell migration in 4T1 and MDA-MB-231 TNBC cells, and ii) inhibiting cell growth in paclitaxel-resistant 4T1 cells (potentiating the paclitaxel antiproliferative effect). Moreover, 20 mg/kg LINS01022 reduced tumor size in 4T1 tumor-bearing mice, exhibiting a safe toxicological profile and potential for druggability estimated by ADME calculations. We conclude that the H₃R is involved in the regulation of TNBC progression, offering promising therapeutic potential for the novel LINS01 series of H₃R antagonists.