Vehicle toxicity is one of the main obstacles for intracellular delivery of bioactive compounds. Silk fibroin is a natural polymer proven to have high biocompatibility since being used as suture material. In this report, fibroin microspheres were prepared without any chemical modification or cross-linking not to affect its biocompatibility. The microspheres were taken up by more than 90% of 3T3 cells. Cellular uptake continued after medium replenishment with a different-colored fluorescent microsphere, suggesting that simultaneous ingestion and exocytosis occurred. Cellular uptake of fibroin microspheres did not affect cell viability. Intracellular trafficking of the microspheres using lysosome-specific fluorescent dye revealed that fibroin microspheres were localized both in the cytoplasm and in the lysosome. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of large cargos, such as mixtures of proteins, nutrients or artificial organelles.