Heat stress (HS) is a major stressor for laying hens. It causes enormous financial losses worldwide annually. The purpose of this study was to investigate the effect and mechanism of a probiotic mixture of Bacillus subtilis and Enterococcus faecium on the performance of laying hens under HS. Eight hundred and fifty-six commercial laying hens (Hy-Line Brown, aged 40 weeks) were randomly allocated to three groups, Group C (26 °C), Group H (33 °C) and Group H + P_M (33 °C + probiotic mixture), respectively. The trial lasted for 20 days. Compared with Group H, we demonstrated that treatment with the probiotic mixture significantly increased the egg production rate, average daily feed intake (ADFI), and egg weight of laying hens under HS (p < .05). In addition, the eggshell thickness, eggshell strength and albumen height also improved. We further showed that these probiotics improved gut microbiota as well as enhanced intestinal integrity, which may be responsible for inhibiting the invasion of bacteria and improving the performance indices. The study suggests that this probiotic mixture may be an effective strategy for treating laying hens under HS.