MicroRNAs (miRNAs) have been the subject of recent attention as key regulatory factors in cell differentiation. In the current study, to explore the early signaling cascade of osteogenic differentiation of human induced pluripotent stem (hiPS) cells, we investigated miR-211 regulation and autophagy-related gene (Atg) signaling in osteogenic differentiation. In addition to reciprocal strong induction of miR-211 expression in differentiated cells following osteogenic differentiation, we found abundant Argonaute 3 bound to miR-211. There were also dramatic increases in the mRNA and protein levels of Atg14 together with increases in the amount of autophagosomes as well as autophagic fluxes. While transfection of a miR-211 inhibitor abrogated the induction of Atg14, autophagy events, osteoblast differentiation markers, and induction of calcification were suppressed markedly. Treatment with small interfering RNAs against Atg14 also suppressed the osteogenic differentiation medium (ODM)-induced increase in osteogenic differentiation. The osteogenic phenotype was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy inducer). Taken together with our previous findings, we have revealed a unique sequential cascade involving miR-211 and Atg14 in ODM-induced differentiation of hiPS cells into osteoblast-like cells at a relatively early stage.