Live-cell imaging is a common technique in microscopy to investigate dynamic cellular behaviour and permits the accurate and relevant analysis of a wide range of cellular and tissue parameters, such as motility, cell division, wound healing responses and calcium (Ca
2
+
) signalling in cell lines, primary cell cultures and
ex vivo
preparations. Furthermore, this can occur under many experimental conditions, making live-cell imaging indispensable for biological research. Systems which maintain cells at physiological conditions outside of a CO
2
incubator are often bulky, expensive and use proprietary components. Here we present an inexpensive, open-source temperature control system for
in vitro
live-cell imaging. Our system ‘ThermoCyte’, which is constructed from standard electronic components, enables precise tuning, control and logging of a temperature ‘set point’ for imaging cells at physiological temperature. We achieved stable thermal dynamics, with reliable temperature cycling and a standard deviation of 0.42°C over 1 h. Furthermore, the device is modular in nature and is adaptable to the researcher's specific needs. This represents simple, inexpensive and reliable tool for laboratories to carry out custom live-cell imaging protocols, on a standard laboratory bench, at physiological temperature.