Objective To analyze the causal relationship between gut microbiota and childhood asthma based on Mendelian randomization (MR). Methods The human gut microbiota dataset was downloaded from the MiBioGen database, and 196 known bacterial groups (9 phyla, 16 classes, 20 orders, 32 families, and 119 genera) were retained as exposure factors. Single nucleotide polymorphisms (SNPs) that were strongly correlated with exposure factors and independent of each other were selected as effective instrumental variables. A childhood asthma dataset with 3 025 patients and 135 449 controls was downloaded from the genome-wide association studies database as the outcome variable. Two-sample MR analysis was performed using inverse variance weighted, weighted median, MR-Egger, weighted model and simple model methods, respectively. The causal association between gut microbiota and childhood asthma was evaluated by odds ratio (OR). Sensitivity analysis was performed by leave-one-out method. Horizontal pleiotropy was tested by MR-Egger intercept test and MR-PRESSO global test, and Cochran’s Q test was used for heterogeneity. Results A total of 15 out of 196 gut microbiota groups were found to have a causal association (P<0.05) with the risk of childhood asthma, with a total of 181 SNPs included in the analysis. Inverse variance weighted analysis showed that Mollicutes [OR=1.42, 95% confidence interval (CI) (1.10, 1.83), P=0.007], Escherichia-Shigella [OR=1.39, 95%CI (1.02, 1.90), P=0.036], Oxalobacter [OR=1.30, 95%CI (1.10, 1.54), P=0.002], Ruminococcaceae UCG-009 [OR=1.34, 95%CI (1.09, 1.64), P=0.006] and Tenericutes [OR=1.42, 95%CI (1.10, 1.83), P=0.007] were significantly positively correlated with childhood asthma. Actinobacteria [OR=0.76, 95%CI (0.58, 0.99), P=0.042], Bifidobacteriaceae [OR=0.76, 95%CI (0.58, 0.98), P=0.035], Eubacterium nodatum group [OR=0.81, 95%CI (0.70, 0.94), P=0.007], Bifidobacterales [OR=0.76, 95%CI (0.58, 0.98), P=0.035] and Actinobacteria [OR=0.74, 95%CI (0.56, 0.99), P=0.040] were negatively correlated with childhood asthma. In addition, the results of leave-one-out sensitivity analysis were stable, MR-Egger intercept test and MR-PRESSO global test showed no horizontal pleiotropy, and Cochran’s Q test showed no heterogeneity. Conclusions There is a causal relationship between gut microbiota and childhood asthma. Mollicutes, Escherichia-Shigella, Oxalobacter, Ruminococcaceae UCG-009 and Tenericutes may increase the risk of childhood asthma. Actinobacteria, Bifidobacteriaceae, Eubacterium nodatum group, Bifidobacterales and Actinobacteria can reduce the risk of childhood asthma.