In vivo calcium imaging is a powerful tool used to record neuronal activity from living animals. For this purpose, two-photon excitation laser-scanning microscopy is commonly used because of the optical accessibility of deep tissues. In this study, we report that one-photon confocal scanning laser microscopy, when optimally tuned, is also applicable for in vivo calcium imaging from the superficial layer of the neocortex. By combining a Nipkow-disk confocal unit with a fluorescence stereo zoom microscope and a high numerical aperture objective, we succeeded in recording the fluorescence signal of individual cells at a depth of up to 160 μm in brain tissues, which corresponds to layer II of the mouse neocortex. In fact, we conducted in vivo functional multineuron calcium imaging and simultaneously recorded spontaneous activity from more than 100 neocortical layer II neurons. This one-photon confocal system provides a simple, low-cost experimental platform for time-lapse imaging from living animals.