HS6ST3 was found up-regulated in T47D, MCF7 and MDA-MB231 breast cancer cell lines. Silencing HS6ST3 diminished tumor cell growth and progression in breast cancer cells. Silencing HS6ST3 down-regulated IGF1R and up-regulated XAF1 in breast cancer cells. Co-targeting IGF1R and XAF1 could reduce the breast cancer growth and progression. Targeting HS6ST3 could be useful in breast cancer therapy.