Advanced technologies for toxicity tests are designed to identify biomarkers with superior predictive power or end points of the complex web of biological pathways. However, the data obtained need to be fully characterized for dose-response, physiological systems, and relevance to a system or (sub) population before biological interpretation and decision making. In this study, the toxicity of triclosan (TCS) on zebrafish was selected as a case study to correlate the observed morphological effects with existing data and identify the critical events by receptor activity sensitivity analysis. Triclosan exhibited weak acute toxicity against zebrafish and significantly affected the development of trunk muscles at 0.52, 1.04, and 1.73 μM. Through receptor-mediated screening, we found that the adverse effects of TCS induce Ryanodine receptor 1 (RyR1) activity and distort Ca2+ signaling. The trunk skeletal muscle abnormalities occurred only when the dihydropyridine receptor (DHPR) was blocked, demonstrating that TCS mainly influenced the Ca2+ regulatory module associated with signaling between DHPRs and RyR1; DHPRs mainly regulated the orthograde and retrograde signaling in skeletal muscles. This unexpected result could integrate the mode of action of TCS and provide insight for high-throughput screening and toxicity prediction using zebrafish.