Breast cancer (BC) is a leading cause of cancer-related deaths among women, with triple-negative breast cancer (TNBC) being particularly aggressive and challenging to treat. MiRNAs are small non-coding RNAs that regulate gene expression. Their aberrant expression is linked to pathologies such as BC. Previously, we found that let-7b-5p was diminished in BC tissue compared to adjacent normal tissue using bioinformatic approaches. The aim of this work was to investigate the effect of let-7b-5p in TNBC. Our hypothesis is that let-7b-5p acts as a tumor suppressor in TNBC. We analyzed let-7b-5p expression levels in PAM50 basal-like BC tumors and normal mammary tissue (NT) from TCGA BRCA and GTEx project data sets. We found that let-7b-5p was significantly diminished in PAM50 basal-like BC tissue compared to NT. We also found that its expression is decreased in basal-like BC compared to the other PAM50 BC molecular subtypes using UCSC Xena tool. Then, we evaluated let-7b-5p effects in the TNBC cells, MDA-MB-231 and 4T1, using stable transfections with expression vectors or transient transfections with a let-7b-5p mimic or negative control (NC), respectively. Overexpression of let-7b-5p reduced the clonogenic capability of MDA-MB-231 cells. On the other hand, let-7b-5p increased the viability under serum deprivation conditions and cell adhesion of 4T1 cells. Moreover, let-7b-5p reduced migration of both, 4T1 and MDA-MB-231 cells. Finally, the effect of treatment with a single dose of PEI nanoparticles containing a let-7b-5p mimic or NC was analyzed in female Balb/c mice with 4T1 orthotopic tumors. Tumor size was decreased on days 11 and 12 after treatment with the let-7b-5p mimic compared to NC. These findings constitute the initial step for developing therapies for TNBC based on a specific let-7b-5p miRNA mimic. Further research is needed to understand the mechanisms by which let-7b-5p influences TNBC and evaluate its effect on metastasis.