Glycoconjugate mucin secretion from conjunctival goblet cells is tightly regulated by nerves and specialized pro-resolving mediators (SPMs) to maintain ocular surface health. Here we investigated the actions of the SPM resolvin E1 (RvE1) on cultured rat conjunctival goblet cell glycoconjugate secretion and intracellular [Ca] ([Ca]) and the signaling pathways used by RvE1. Goblet cells were cultured from rat conjunctiva in RPMI medium. The amount of RvE1-stimulated glycoconjugate mucin secretion was determined using an enzyme-linked lectin assay with Ulex Europaeus Agglutinin 1 lectin. Cultured goblet cells were also incubated with the Ca indicator dye fura 2/AM and [Ca] was measured. Cultured goblet cells were incubated with inhibitors to phospholipase (PL-) C, D, and A2 signaling pathways. RvE1 stimulated glycoconjugate secretion in a concentration dependent manner and was inhibited with the Ca chelator BAPTA. The Ca response was also increased in a concentration manner when stimulated by RvE1. Inhibition of PLC, PLD, and PLA2, but not Ca/calmodulin-dependent kinase blocked RvE1-stimulated increase in [Ca] and glycoconjugate secretion. We conclude that under normal, physiological conditions RvE1 stimulates multiple pathways to increase glycoconjugate secretion and [Ca]. RvE1 could be an important regulator of goblet cell glycoconjugate mucin secretion to maintain ocular surface health.