Operating temperature of Lithium-ion batteries (LIBs) significantly affects their electrochemical-thermal performance, cycle life, and cost. This study presents the thermal and electrochemical performance of 20 Ah LiFePO4 cells for 8 current rates (0.2C–5C) at 8 operating temperatures (−20 °C to 50 °C). Results show that the effects of operating temperature and current rate on cell performance differ above 10 °C, between 10 °C and 0 °C, and at subzero temperatures. Based on the electrochemical impedance spectroscopy (EIS) measurements, significantly higher bulk and charge-transfer resistances in conjunction with the lower diffusion coefficients results in poor battery efficiency at subzero temperatures. Optimum operating condition is 50 °C at a rate of 0.2C, in terms of utilized power and capacity, while a considerable power loss and capacity decrease occur below 20 °C. Furthermore, increasing the current rate is detrimental above 0 °C, whereas it improves cell performance at −10 °C, in terms of cell capacity. Moreover, cell temperature reaches an undesirable value at 50 °C and 5C rate, thus a thermal management system is necessary for high capacity LiFePO4 cells at higher temperatures and/or at higher C-rates. Additionally, temperature differences on the surface of high capacity cells reach 10 °C below room temperature at high current rates which can lead to non-uniform material utilization, and consequently cell failures. Finally, the cycle life of 20 Ah LiFePO4 cells decreases dramatically as discharge current rate increases.