Individual cells play essential roles in the biological processes of the brain. The number of neurons changes during both normal development and disease progression. High-resolution imaging has made it possible to directly count cells. However, the automatic and precise segmentation of touching cells continues to be a major challenge for massive and highly complex datasets. Thus, an integrative cut (iCut) algorithm, which combines information regarding spatial location and intervening and concave contours with the established normalized cut, has been developed. iCut involves two key steps: (1) a weighting matrix is first constructed with the abovementioned information regarding the touching cells and (2) a normalized cut algorithm that uses the weighting matrix is implemented to separate the touching cells into isolated cells. This novel algorithm was evaluated using two types of data: the open SIMCEP benchmark dataset and our micro-optical imaging dataset from a Nissl-stained mouse brain. It has achieved a promising recall/precision of 91.2 ± 2.1%/94.1 ± 1.8% and 86.8 ± 4.1%/87.5 ± 5.7%, respectively, for the two datasets. As quantified using the harmonic mean of recall and precision, the accuracy of iCut is higher than that of some state-of-the-art algorithms. The better performance of this fully automated algorithm can benefit studies of brain cytoarchitecture.