Slits are a group of secreted glycoproteins that act as molecular guidance cues in cellular migration. Recently, several studies demonstrated that Slit-2 can operate as candidate tumour suppressor protein in various tissues. In this study, we show Slit-2 expression in basal cell layers of normal oral mucosa colocalized with P-cadherin expression. In contrast, there is a loss of Slit-2 and P-cadherin expression in mucosa of oral squamous cell carcinoma (OSCC). Our in vitro investigations reveal a correlation of P-cadherin and Slit-2 expression: OSCC cells with induced P-cadherin expression (PCI52_PC) display an increased Slit-2 expression. However, abrogating P-cadherin function with a function-blocking antibody decreases Slit-2 secretion confirming a direct link between P-cadherin and Slit-2. Moreover, experiments with OSCC cells show that Slit-2 interferes with a Wnt related signalling pathway, which in turn affects Slit-2 expression in a feedback loop. Functionally, transwell migration assays demonstrate a Slit-2 dose-dependent decrease of PCI52_PC cell migration. However, there is no influence on migration in mock control cells. Responsible for this migration block might be an interaction of P-cadherin with Roundabout (Robo)-3, a high affinity receptor of Slit-2. Indeed, proximity ligation assays exhibit P-cadherin/Robo-3 interactions on PCI52_PC cells. Additionally, we detect a modulation of this interaction by addition of recombinant Slit-2. Down-regulation of Robo-3 expression via small interfering RNA neutralizes Slit-2 induced migration block in PCI52_PC cells. In summary, our experiments show antitumorigenic effects of Slit-2 on P-cadherin expressing OSCC cells supposedly via modulation of Robo-3 interaction.