Folate receptor (FR) is highly expressed in many types of human cancers, and it has been actively studied for developing targeted chemotherapy and diagnostic agents. Tremendous efforts have been made in developing FR-targeted nanomedicines and nanoprobes and translating them into clinical applications. This article provides a concise review on the latest development of folate-mediated nanomedicines and nanoprobes for chemotherapy and diagnostics with an emphasis on in vivo applications. The cellular uptake mechanisms, pharmacokinetics (PK), administration routes and major challenges in FR-targeted nanoparticles are discussed.