Human-induced environmental change can affect the evolutionary trajectory of populations. In Mexico, indigenous Zoque people annually introduce barbasco, a fish toxicant, into the Cueva del Azufre to harvest fish during a religious ceremony. Here, we investigated tolerance to barbasco in fish from sites exposed and unexposed to the ritual. We found that barbasco tolerance increases with body size and differs between the sexes. Furthermore, fish from sites exposed to the ceremony had a significantly higher tolerance. Consequently, the annual ceremony may not only affect population structure and gene flow among habitat types, but the increased tolerance in exposed fish may indicate adaptation to human cultural practices in a natural population on a very small spatial scale.