Prostate cancer (PC) is one of the most common cancers among men, and vitamin D and its metabolites are candidates for prevention and therapy of this disease. The vitamin D metabolites, 1, 25-dihydroxyvitamin D3 (1,25D) and 25-hydroxyvitamin D3, decreases cellular proliferation and invasiveness, and stimulates differentiation of PC cells. However, the underlying mechanisms are not fully clarified, and there is evidence that some of these effects of the vitamin D system are mediated by specific membrane-associated receptors/binding proteins in addition to its nuclear receptor, suggesting multiple regulatory pathways. The aim of the present study was to examine the role of membrane initiated pathways mediating effects of 1,25D on cell invasiveness in LNCaP cells. Treatment with 1,25D evoked a dose-dependent activation of the JNK/SAPK MAPK signaling pathways within 10 min, demonstrating membrane initiated signaling of 1,25D in LNCaP cells. Furthermore, treatment with 1,25D decreased LNCaP cell invasiveness by approximately 20% after 48 h. Using an inhibitor (SP600125) for the JNK/SAPK MAPK signaling pathway in combination with 1,25D on LNCaP cells, the inhibitory action of 1,25D on invasiveness was eliminated. In conclusion, 1,25D decrease invasiveness of LNCaP cells by interaction with a putative membrane associated receptor, which activate membrane, initiated signaling via the JNK/SAPK MAPK signaling pathway.