Probiotics are important biological control products in the aquaculture of sea cucumber, but probiotics used specifically in low‐temperature environments are rare. The present study used treatment with low‐temperature‐resistant Sporosarcina aquimarina MS4 and investigated changes in growth, immunity, intestinal flora and immune‐related gene expression in sea cucumber at low water temperature (8°C). After 60 days of feeding, S. aquimarina MS4 significantly improved growth performance and immune enzyme activity and reduced mortality in sea cucumbers infected with Vibrio splendidus compared with the commercial diet. The intestinal flora structure of the sea cucumbers fed S. aquimarina MS4 differed from that of the control group. Compared with that of the control group, the intestinal flora of sea cucumbers fed S. aquimarina MS4 was always dominated by Proteobacteria and had a higher abundance of Verrucobacteria, and the flora structure was more stable. The expression of the TLR, MyD88, IRAK4, p38 and NFκB genes was upregulated significantly after the addition of S. aquimarina MS4 to the diet. In summary, low‐temperature‐resistant S. aquimarina MS4 could regulate the intestinal flora and immune‐related gene expression of sea cucumber, thereby improving its immunity and resistance to pathogenic bacteria such as V. splendidus.