Osterix/Sp7 is a zinc finger transcription factor and critical regulator of osteoblast differentiation, maturation and activity. Osterix expression has also been described in non-skeletal tissues but functional analyses are lacking. In the present study, we show that in the teleost model medaka, osterix is present as two alternatively spliced transcripts, osx_tv1 and osx_tv2. Knock-down of osx_tv1 and/or osx_tv2 results in mineralization loss in early intramembranous bones while cartilage formation is mostly unaffected. Formation of the parasphenoid, the earliest mineralized bone in the medaka skeleton, is impaired and fails to recover at later stages. Ossification of later bones, such as the operculum and cleithrum, is delayed but recovers during further development. In the axial skeleton, formation of the neural arches and centra is strongly delayed. In vivo analyses using osterix:nlGFP and osteocalcin:GFP transgenic medaka and whole mount in situ hybridization suggest that bone defects observed after knock-down of osterix are caused by a delay of osteoblast maturation and activity. Furthermore, we analyzed expression profile and function of osterix during ear and otolith formation. We show that osterix is expressed in otic placodes at the otic vesicle stage and that its knock-down results in a loss of otoliths. Taken together, we show that osterix is required for bone formation in a teleost fish and that its important regulatory functions are conserved between teleosts and mammals. Furthermore, we provide the first functional evidence for a role of Osterix in a non-skeletal tissue, i.e. the otoliths.