Heparanase is the sole mammalian enzyme capable of cleaving heparan sulfate, a glycosaminoglycan that constitutes a major component of the extracellular matrix and cell surfaces. Heparanase has previously been implicated in immune cell function, however, its precise role in natural killer (NK) cell activation and function remains to be well defined. In this study, we challenged heparanase-deficient (Hpse-/-) mice with polyinosinic:polycytidylic acid (poly(I:C)) and found Hpse-/- NK cell cytotoxicity was impaired against EO771.LMB mouse breast cancer cells. Hpse-/- NK cells also expressed lower levels of the activation markers CD69 and NKG2D than wild-type NK cells after in vivo poly(I:C) stimulation. In vitro stimulation of NK cells with IL-12 and IL-15, or IL-12 and IL‑18 induced the expression of CD69 and NKG2D equally in wild-type and Hpse‑/- NK cells, however, Hpse-/- NK cells produced less monocyte chemoattractant protein-1 (MCP‑1). NK cells are activated in vivo by dendritic cells (DCs) in response to poly(I:C). Hence, we investigated the role of heparanase in DC-mediated NK cell activation. Poly(I:C)-stimulated Hpse‑/- bone marrow DCs (BMDCs) expressed less IL‑12 than wild-type BMDCs, an important cytokine in NK cell activation. Also, when poly(I:C)-stimulated BMDCs were cultured with Hpse-/- NK cells, less MCP-1 mRNA and protein were expressed than in wild-type controls. Cell-cell contact is important for DC-mediated NK cell activation. However, co-cultures of Hpse-/- BMDCs and Hpse-/- NK cells showed similar levels of cell-cell contact, in frequency and duration, to wild-type conditions. These results demonstrate that heparanase is important for DC-mediated NK cell cytotoxicity and activation in vivo, and for NK cell-expression of MCP‑1. These results also suggest that the role of heparanase in DC-mediated NK cell activation occurs independently of cell-cell contact. These findings raise the question of how heparanase inhibitors, currently in development as anti-cancer therapies, may affect immune surveillance and uncovers a new approach to increasing immune cell-crosstalk in immunotherapies.