Diacylglycerol (DAG) is required for membrane traffic and structural organization at the Golgi. DAG is a lipid metabolite of several enzymatic reactions present at this organelle, but the mechanisms by which they are regulated are still unknown. Here, we show that cargo arrival at the Golgi increases the recruitment of the DAG-sensing constructs C1-PKCθ-GFP and the PKD-wt-GFP. The recruitment of both constructs was reduced by PLCγ1 silencing. Post-Golgi trafficking of transmembrane and soluble proteins was impaired in PLCγ1-silenced cells. Under basal conditions, PLCγ1 contributed to the maintenance of the pool of DAG associated with the Golgi and to the structural organization of the organelle. Finally, we show that cytosolic phospholipase C (PLC) can hydrolyse phosphatidylinositol 4-phosphate in isolated Golgi membranes. Our results indicate that PLCγ1 is part of the molecular mechanism that couples cargo arrival at the Golgi with DAG production to co-ordinate the formation of transport carriers for post-Golgi traffic.