Heparan sulfate 6-O-sulfation is biologically edited by 6-O-sulfotransferases (HS6STs) within heparan sulfate chains. Three isoforms of HS6ST have been identified. These enzymes are found to be differentially expressed in a variety of tissues. Recently, several studies have shown that dysregulation of 6-O-sulfotransferases could be involved in tumorigenesis of several cancers. This study aimed to analyze the expression and function of HS6ST3 in breast cancer. HS6ST3 was found up-regulated in T47D, MCF7 and MDA-MB231 breast cancer cell lines. HS6ST3 was then silenced in T47D and MCF7 using siRNA. Silencing HS6ST3 diminished tumor cell growth, migration and invasion, but enhanced cell adhesion and apoptosis in breast cancer. Gene microarray analysis revealed that silencing HS6ST3 significantly changed the expression of IGF1R and XAF1 in breast cancer cells. Further functional studies showed that the cellular processes were mediated by IGF1R and XAF1 after silencing HS6ST3 in breast cancer cells. Together these results indicate that HS6ST3 might be involved in the tumorigenesis of breast cancer and it could be a promising target in breast cancer therapy.