PilZ domain-containing proteins constitute a large family of bacterial signaling proteins. As a widely distributed protein domain for the binding of the second messenger c-di-GMP, the canonical PilZ domain contains a set of motifs that define the binding site for c-di-GMP and an allosteric switch for propagating local conformational changes. Here, we summarize some new insights gathered from recent studies on the commonly occurring single-domain PilZ proteins, YcgR-like proteins and PilZ domain-containing cellulose synthases. The studies collectively illuminate how PilZ domains function as cis- or trans-regulatory domains that enable c-di-GMP to control the activity of its cellular targets. Overall, the review highlights the diverse protein structure, biological function and regulatory mechanism of PilZ domain-containing proteins, as well as the challenge of deciphering the function and mechanism of orphan PilZ proteins.