The aim of this study was to identify genes and molecular pathways differentially regulated by synchrotron-generated microbeam radiotherapy (MRT) versus conventional broadbeam radiotherapy (CRT) in vitro using cultured EMT6.5 cells. We hypothesized (based on previous findings) that gene expression and molecular pathway changes after MRT are different from those seen after CRT. We found that at 24 h postirradiation, MRT exerts a broader regulatory effect on multiple pathways than CRT. MRT regulated those pathways involved in gene transcription, translation initiation, macromolecule metabolism, oxidoreductase activity and signaling transduction in a different manner compared to CRT. We also found that MRT/CRT alone, or when combined with inflammatory factor lipopolysaccharide, upregulated expression of Ccl2, Ccl5 or Csf2, which are involved in host immune cell recruitment. Our findings demonstrated differences in the molecular pathway for MRT versus CRT in the cultured tumor cells, and were consistent with the idea that radiation plays a role in recruiting tumor-associated immune cells to the tumor. Our results also suggest that a combination of MRT/CRT with a treatment targeting CCL2 or CSF2 could repress the tumor-associated immune cell recruitment, delay tumor growth and/or metastasis and yield better tumor control than radiation alone.