Quantum dots (QDs) have size-dependent optical properties that make them uniquely advantageous for in vivo targeted fluorescence imaging, traceable delivery, and therapy. The use of group II-VI (e.g., CdSe) QDs for these applications is advancing rapidly. However, group II-VI QDs contain toxic heavy metals that limit their in vivo applications. Thus, replacing these with QDs of a biocompatible semiconductor, such as silicon (Si), is desirable. Here, we demonstrate that properly encapsulated biocompatible Si QDs can be used in multiple cancer-related in vivo applications, including tumor vasculature targeting, sentinel lymph node mapping, and multicolor NIR imaging in live mice. This work overcomes dispersibility and functionalization challenges to in vivo imaging with Si QDs through a unique nanoparticle synthesis, surface functionalization, PEGylated micelle encapsulation, and bioconjugation process that produces bright, targeted nanospheres with stable luminescence and long (>40 h) tumor accumulation time in vivo. Upon the basis of this demonstration, we anticipate that Si QDs can play an important role in more sophisticated in vivo models, by alleviating QD toxicity concerns while maintaining the key advantages of QD-based imaging methods.