The few existing studies on deep-sea hydrothermal vent copepods indicate low connectivity with surrounding environments and reveal high endemism among vents. However, the finding of non-endemic copepod species in association with engineer species at different reduced ecosystems poses questions about the dispersal of copepods and the colonization of hydrothermal vents as well as their ecological connectivity. The objective of this study is to understand copepod colonization patterns at a hydrothermal vent site in response to environmental factors such as temperature and fluid flow as well as the presence of different types of substrata. To address this objective, an in situ experiment was deployed using both organic (woods, pig bones) and inorganic (slates) substrata along a gradient of hydrothermal activity at the Lucky Strike vent field (Eiffel Tower, Mid-Atlantic Ridge). The substrata were deployed in 2011 during the MoMARSAT cruise and were recovered after two years in 2013. Overall, copepod density showed significant differences between substrata types, but was similar among different hydrothermal activity regimes. Highest densities were observed on woods at sites with moderate or low fluid input, whereas bones were the most densely colonized substrata at the 2 sites with higher hydrothermal influence. Although differences in copepod diversity were not significant, the observed trends revealed overall increasing diversity with decreasing temperature and fluid input. Slates showed highest diversity compared to the organic substrata. Temperature and fluid input had a significant influence on copepod community composition, resulting in higher similarity among stations with relatively high and low fluid inputs, respectively. While vent-specialists such as dirivultids and the tegastid Smacigastes micheli dominated substrata at high vent activity, the experiment demonstrated increasing abundance and dominance of non-vent taxa with decreasing temperature and fluid input. Effects of the substratum type on community composition were not significant, although at sites with moderate or low fluid input, woods exhibited distinctive communities with high densities and relative abundance of the taxon Nitocrella sp.. In conclusion, copepod colonization and species composition were mainly influenced by hydrothermal fluid input and temperature rather than the type of substratum. The outcome of this study provides fundamental knowledge to better understand copepod colonization at hydrothermal vents