Taspase1, a highly conserved threonine protease, cleaves nuclear transcriptional regulators mixed-lineage leukemia (MLL, MLL1), MLL2, TFIIA, and ALF to orchestrate a wide variety of biological processes. In vitro studies thus far demonstrated that Taspase1 plays important roles in the proliferation of various cancer cell lines, including HER2-positive breast cancer cells. To investigate the role of Taspase1 in breast tumorigenesis in vivo, we deleted Taspase1 from mouse mammary glands by generating MMTV-neu; MMTV-cre; Tasp1 F/− mice. We demonstrate that initiation of MMTV-neu-but not MMTV-wnt-driven breast cancer is blocked in the absence of Taspase1. Importantly, Taspase1 loss alone neither impacts normal development nor pregnancy physiology of the mammary gland. In mammary glands Taspase1 deficiency abrogates MMTV-neu-induced cyclins E and A expression, thereby preventing tumorigenesis. The mechanisms were explored in HER2-positive breast cancer cell line BT474 and HER2-transformed MCF10A cells and validated using knockdown-resistant Taspase1. As Taspase1 was shown to cleave MLL which forms complexes with E2F transcription factors to regulate Cyclins E, A, and B expression in mouse embryonic fibroblasts (MEFs), we investigated whether the cleavage of MLL by Taspase1 constitutes an essential in vivo axis for HER2/neu-induced mammary tumorigenesis. To this end, we generated MMTV-neu; MLL nc/nc transgenic mice that carry homozygous non-cleavable MLL alleles. Remarkably, these mice are also protected from HER2/neu-driven breast tumorigenesis. Hence, MLL is the primary Taspase1 substrate whose cleavage is required for MMTV-neu-induced tumor formation. As Taspase1 plays critical roles in breast cancer pathology, it may serve as a therapeutic target for HER2-positive human breast cancer.