The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca2+, initiated by the release of Ca2+ from the sarcoplasmic reticulum and terminated by re-sequestration of Ca2+ into the sarcoplasmic reticulum as the main mechanism of Ca2+ removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN)1,2. Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca2+ enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca2+-ATPase in a previously undescribed E1 state, with exposure of the Ca2+ sites through an open cytoplasmic pathway stabilized by Mg2+. The structure suggests a mechanism for selective Ca2+ loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca2+. These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.