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Neophobic predator avoidance (NPA), the fear response exhibited by prey animals exposed to novel stimuli, is a plastic trait thought to be induced by exposure to elevated chronic risk of predation. Indeed, prey experiencing low levels of background risk fail to display NPA, while those experiencing high background risk do display NPA. Recent work has suggested that the trigger for inducing and maintaining NPA may not be background risk per se but rather uncertainty in the predation environment. Here, we designed two experiments on wild-caught neophobic Trinidadian guppies (Poecilia reticulata) to test the uncertainty hypothesis'. In the first experiment, we tested if the diversity of novel stimuli encountered by guppies would affect the maintenance of their NPA. We found that exposure to a single novel odour once per day for 3days was sufficient to extinguish the neophobic response to that odour (Experiment 1A) but not a new odour (Experiment 1B). However, when guppies were exposed to multiple novel odours so that each odour was encountered once a day for 3days, they retained their NPA to both a previously encountered novel odour (Experiment 1A) and a new odour (Experiment 1B). In our second experiment, we repeatedly exposed guppies to a novel cue in a spatially predictable vs. unpredictable location and found that spatial unpredictability interfered with the reduction of NPA to that cue. Combined, our results suggest that uncertainty of risk experienced by prey in time and space shapes the retention of NPA.Significance statementWhen prey face uncertainty, or lack of complete information, about risk in their environment, they engage in adaptive antipredator behaviours such as neophobic predator avoidance. Showing a fear response to a novel stimulus in a high-risk environment is likely adaptive if we assume that most species in that environment are threats. Here, we aim to show how uncertainty, as opposed to simply background levels of risk, may influence the maintenance of neophobic responses in prey. On the basis of two experiments, we showed that prey experiencing increased uncertainty, via increased diversity and spatial unpredictability of novel cues, maintain their neophobic responses more so than prey experiencing lower uncertainty. Understanding the effects of ecological uncertainty on prey behaviour, particularly neophobia, will shed light on how prey might respond as uncertainty increases with climate change, invasive species and anthropogenic habitat modification.