microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1’s function during development. Display omitted •Sea urchin miR-1 has broad expression and function in the developing embryo.•miR-1 regulates gut muscle structures.•miR-1 regulates skeletogenic cell patterning and skeletal spicules.•miR-1 is likely to directly suppress skeletogenic gene regulatory components. This study identifies miR-1 to regulate circumpharyngeal muscle structure, skeletal branching, and skeletal cell patterning. Using site directed mutagenesis and reporter constructs, we identified that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, Notch, and Wnt1 signaling components.