Background: AHNAK, also known as desmoyokin, is a giant protein with the molecular size of approximately 700 kDa and exerts diverse functions in different types of cancer. Results: In the present study, we demonstrated that AHNAK mRNA levels were down-regulated in 7 out of 8 human breast cancer cell lines, especially in triple - negative breast cancer (TNBC) cell lines. Moreover, in patients with TNBC, the expression of AHNAK gene was inversely correlated with the tumor status (P = 0.015), lymph node status (P < 0.001), lymph node (LN) infiltration (P < 0.001) and TNM stage (P < 0.001). Moreover, down-regulated AHNAK expression was considered an independent prognostic factor associated with the poor survival of patients with TNBC. Overexpression of AHNAK in two TNBC cell lines, MDA-MB-231 and BT549, suppressed the in vitro TNBC cell proliferation and colony formation, and inhibited the in vivo TNBC xenograft growth and lung metastasis. The tumor suppressing effect of AHNAK in TNBC was associated with the AKT/MAPK signaling pathway and Wnt/beta-catenin pathway. Consistent results were observed when AHNAK was knockdown in BT20 and MDA-MB-435 cells. Conclusions: Taken together, our results suggest that AHNAK acts as a tumor suppressor that negatively regulates TNBC cell proliferation, TNBC xenograft growth and metastasis via different signaling pathways.