TWIST1, Slug, Snail, SIP1, and NF-κB are overexpressed in various tumors and associated with metastasis and poor prognosis. In this study, we examined their potential roles in phyllodes tumor (PT). The expression of TWIST1, Snail, Slug, SIP1, and NF-κB in benign (n = 103), borderline (n = 38), and malignant (n = 38) PTs was examined by immunostaining. The methylation status of the TWIST1 promoter was analyzed by methylation-specific PCR. We detected high expression levels of TWIST1 in 47.4 % of borderline/malignant PTs and 31.1 % of benign PTs, Slug in 64 % of borderline/malignant PTs and 62.1 % of benign PTs, epithelial SIP1 in 75.0 % of borderline/malignant PTs and 86.3 % of benign PTs, stromal SIP1 in 35.5 % of borderline/malignant PTs and 22.3 % of benign PTs, and NF-κB in 63.2 % of borderline/malignant PTs and 52.4 % of benign PTs. Snail expression was detected in all cases. A high expression of TWIST1 (p = 0.026) and TWIST1 promoter methylation (p = 0.000) were significantly more frequent in borderline/malignant PTs than in benign PTs. Moreover, a high expression of at least four of the five antibodies was more commonly observed in borderline/malignant PTs than in benign PTs (p = 0.026). However, no relationship was found between the expression of TWIST1 or the other proteins examined and the clinical outcome. Our results suggest that a high expression of TWIST1 and related proteins plays a pivotal role in the malignant progression of PT.