Protein secretion systems are crucial tools that microbes use to interact with their surrounding environment. The type VI protein secretion system (T6SS) is widely distributed in Gram-negative bacteria and capable of delivering effector to both eukaryotic and prokaryotic cells. Here we discuss, from effector study perspective, the mechanism of substrate recognition and translocation by T6SS, focusing on the key discoveries of VgrG-PAAR (proline-alanine-alanine-aRginine) dependent secretion pathway, and its cognate effectors and chaperone proteins.