During the last two decades, Bacillus megaterium has been systematically developed for the gram-per-liter scale production of recombinant proteins. The plasmid-based expression systems employed use a xylose-controlled promoter. Protein production analyses at the single cell level using green fluorescent protein as a model product revealed a cell culture heterogeneity characterized by a significant proportion of low-producing bacteria. Due to the enormous size of B. megaterium, such bistable behavior seen in subpopulations was readily analyzed by time-lapse microscopy and flow cytometry. Cell culture heterogeneity was not simply caused by plasmid loss: Instead, an asymmetric distribution of plasmids during cell division was detected during the exponential growth phase. Multi-copy plasmids are generally randomly distributed between daughter cells. However, in vivo and in vitro experiments demonstrated that under conditions of strong protein production, plasmids are retained at one of the cell poles. Furthermore, it was found that cells with accumulated plasmids and high protein production ceased cell division. As a consequence, the overall protein production of the culture was mainly achieved by the subpopulation with a sufficient plasmid copy number. Based on our experimental data, we propose a model whereby the distribution of multi-copy plasmids is controlled by polar fixation under protein production conditions. Thereby, cell lines with fluctuating plasmid abundance arise, which results in population heterogeneity. Our results provide initial insights into the mechanism of cellular heterogeneity during plasmid-based recombinant protein production in a Bacillus species.