We have previously demonstrated that crosstalk between lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) facilitates breast cancer proliferation. However, the underlying mechanisms are largely unknown. Here, we report that expression of HDAC5 and LSD1 proteins were positively correlated in human breast cancer cell lines and tissue specimens of primary breast tumors. Protein expression of HDAC5 and LSD1 was significantly increased in primary breast cancer specimens in comparison with matched-normal adjacent tissues. Using HDAC5 deletion mutants and co-immunoprecipitation studies, we showed that HDAC5 physically interacted with the LSD1 complex through its domain containing nuclear localization sequence and phosphorylation sites. Although the in vitro acetylation assays revealed that HDAC5 decreased LSD1 protein acetylation, small interfering RNA (siRNA)-mediated HDAC5 knockdown did not alter the acetylation level of LSD1 in MDA-MB-231 cells. Overexpression of HDAC5 stabilized LSD1 protein and decreased the nuclear level of H3K4me1/me2 in MDA-MB-231 cells, whereas loss of HDAC5 by siRNA diminished LSD1 protein stability and demethylation activity. We further demonstrated that HDAC5 promoted the protein stability of USP28, a bona fide deubiquitinase of LSD1. Overexpression of USP28 largely reversed HDAC5-KD-induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein. Depletion of HDAC5 by shRNA hindered cellular proliferation, induced G1 cell cycle arrest, and attenuated migration and colony formation of breast cancer cells. A rescue study showed that increased growth of MDA-MB-231 cells by HDAC5 overexpression was reversed by concurrent LSD1 depletion, indicating that tumor-promoting activity of HDAC5 is an LSD1 dependent function. Moreover, overexpression of HDAC5 accelerated cellular proliferation and promoted acridine mutagen ICR191-induced transformation of MCF10A cells. Taken together, these results suggest that HDAC5 is critical in regulating LSD1 protein stability through post-translational modification, and the HDAC5-LSD1 axis has an important role in promoting breast cancer development and progression.