Abstract Background The most common toxic side effect after chemotherapy, one of the main treatments for colorectal cancer (CRC), is myelosuppression. Objective To analyze the correlation between gut microbiota and leukopenia after chemotherapy in CRC patients. Methods Stool samples were collected from 56 healthy individuals and 55 CRC patients. According to the leukocytes levels in peripheral blood, the CRC patients were divided into hypoleukocytes group (n = 13) and normal leukocytes group (n = 42). Shannon index, Simpson index, Ace index, Chao index and Coverage index were used to analyze the diversity of gut microbiota. LDA and Student's t-test(St test) were used for analysis of differences. Six machine learning algorithms, including logistic regression (LR) algorithm, random forest (RF) algorithm, neural network (NN) algorithm, support vector machine (SVM) algorithm, catboost algorithm and gradient boosting tree algorithm, were used to construct the prediction model of gut microbiota with leukopenia after chemotherapy for CRC. Results Compared with healthy group, the microbiota alpha diversity of CRC patients was significantly decreased (p