ABSTRACTCholera has been a human scourge since the early 1800s and remains a global public health challenge, caused by the toxigenic strains of the bacteriumVibrio cholerae. In its aquatic reservoirs,V. choleraehas been shown to live in association with various arthropod hosts, including the chironomids, a diverse insect family commonly found in wet and semiwet habitats. The association betweenV. choleraeand chironomids may shield the bacterium from environmental stressors and amplify its dissemination. However, the interaction dynamics betweenV. choleraeand chironomids remain largely unknown.  In this study, we developed freshwater microcosms with chironomid larvae to test the effects of cell density and strain onV. cholerae-chironomid interactions. Our results show that chironomid larvae can be exposed toV. choleraeup to a high inoculation dose (109cells/mL) without observable detrimental effects. Meanwhile, interstrain variability in host invasion, including prevalence, bacterial load, and effects on host survival, was highly cell density-dependent. Microbiome analysis of the chironomid samples by 16S rRNA gene amplicon sequencing revealed a general effect ofV. choleraeexposure on microbiome species evenness. Taken together, our results provide novel insights intoV. choleraeinvasion dynamics of the chironomid larvae with respect to various doses and strains. The findings suggest that aquatic cell density is a crucial driver ofV. choleraeinvasion success in chironomid larvae and pave the way for future work examining the effects of a broader dose range and environmental variables (e.g., temperature) onV. cholerae-chironomid interactions.IMPORTANCEVibrio choleraeis the causative agent of cholera, a significant diarrheal disease affecting millions of people worldwide. Increasing evidence suggests that the environmental facets of theV. choleraelife cycle involve symbiotic associations with aquatic arthropods, which may facilitate its environmental persistence and dissemination. However, the dynamics of interactions betweenV. choleraeand aquatic arthropods remain unexplored. This study capitalized on using freshwater microcosms with chironomid larvae to investigate the effects of bacterial cell density and strain onV. cholerae-chironomid interactions. Our results suggest that aquatic cell density is the primary determinant ofV. choleraeinvasion success in chironomid larvae, while interstrain variability in invasion outcomes can be observed under specific cell density conditions. We also determined thatV. choleraeexposure generally reduces species evenness of the chironomid-associated microbiome. Collectively, these findings provide novel insights intoV. cholerae-arthropod interactions using a newly developed experimental host system.