Cannabinoid receptors (CBRs) belong to G protein-coupled receptor superfamily, which activation in salivary cells inhibits agonist-stimulated salivation and modifies saliva content. However, the role of different CBR subtypes in acinar cell physiology and in intracellular signalling remains unclear. Here, we uncover the functioning of CB1Rs and CB2Rs in acinar cells of rat submandibular gland and their essential role in saliva secretion. Pharmacological activation of CB1Rs and CB2Rs in the submandibular gland suppressed saliva outflow and modified saliva content produced by the submandibular gland in vivo. Using Na+-selective microelectrodes to record secretory Na+ responses in the lumen of acini we observed a reduction in Na+ transport following the activation of CBRs, which was counteracted by the selective CB1Rs antagonist, AM251. In addition, activation of CB1Rs or CB2Rs caused inhibition of Na+-K+-ATPase activity in microsomes derived from the gland tissue as well as isolated acinar cells. Using Ca2+ imaging technique, we showed that an activation of both CB1Rs and CB2Rs alters [Ca2+]cyt signalling in acinar cells by distinct pathways, involving Ca2+ release from the endoplasmic reticulum (ER) and store-operated Ca2+ entry (SOCE), respectively. Our data demonstrate the functional expression of CB1Rs and CB2Rs in acinar cells, and their implications in the regulation of salivary gland functioning.