PURPOSE: This study investigates whether post-chemotherapeutic use of live and heat-killed Lactobacillus rhamnosus GG can modulate the expression of three pro-inflammatory cytokines in 5-fluorouracil (5-FU)-induced intestinal mucositis in vitro. METHODS: Live L. rhamnosus GG and heat-killed L. rhamnosus GG were observed using scanning electron microscopy. To establish the duration required for optimal expression of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-12 (IL-12), 5 μM of 5-FU was selected to treat 10-day-old Caco-2 cells for 4, 6, 8, and 24 h. Caco-2 cells were treated with 5-FU (5 μM) for 4 h, followed by the administration of live L. rhamnosus GG (multiplicity of infection = 25), and heat-killed L. rhamnosus GG for 2 and 4 h. Finally, total cellular RNA was isolated to quantify mRNA expression of TNF-α, MCP-1, and IL-12 using real-time PCR. RESULTS: The results demonstrated that heat-killed L. rhamnosus GG remained structurally intact with elongation. A biphasic upregulated expression of TNF-α, MCP-1, and IL-12 was observed in 5-FU-treated Caco-2 cells at 4 and 24 h. Compared to non-L. rhamnosus GG controls in 5-FU-pretreated Caco-2 cells, a 2-h treatment of heat-killed L. rhamnosus GG significantly upregulated the MCP-1 expression (p < 0.05), and both live and heat-killed L. rhamnosus GG treatments lasting 4 h upregulated the TNF-α and MCP-1 expression (p < 0.05). Only live L. rhamnosus GG upregulated the IL-12 expression (p < 0.05). CONCLUSIONS: Post-chemotherapeutic use of live or heat-killed L. rhamnosus GG can upregulate the gene expression of 5-FU-induced pro-inflammatory cytokines in Caco-2 cells. Human intestinal epithelium may be vulnerable to the post-chemotherapeutic use of L. rhamnosus GG in 5-FU-induced mucositis that requires further in vivo studies for clarification.