Dynamic charge carriers play a vital role in active photonic quantum/nanodevices, such as electrically pumped semiconductor lasers. Here we present a systematic experimental study of gain‐providing charge‐carrier distribution in a lasing interband cascade laser. The unique charge‐carrier distribution profile in the quantum‐well active region is quantitatively measured at nanometer scales by using a noninvasive scanning voltage microscopy technique. Experimental results clearly confirm the accumulation and spatial segregation of holes and electrons in the beating heart of the device. The measurement also shows that the charge‐carrier density is essentially clamped in the presence of stimulated emission at low temperatures. The threshold charge‐carrier density exhibits a linear but fairly weak temperature dependence, in contrast to the exponential temperature dependence of the threshold current. The experimental approach will lead to a deeper understanding of fundamental processes that govern the operation and performance of nanoelectronic devices, quantum devices and optoelectronic devices.