Sterols are essential nutrients for bees which are thought to obtain them exclusively from pollen. It is possible that variability in pollen sterol content shapes pollinator-flower relationships, as bee species require the physiological capacity or behavioral adaptations to cope with unfavorable sterolic composition of pollen. One behavioral adaptation used by generalist bees to avoid deficiencies is the mixing of different pollen types from multiple botanical families to achieve an optimal nutritional balance. However, a possible strategy that has never been investigated is the specific addition of nutrients by adult bees to pollen during foraging trips. Here, we analyzed the pollen sterols from 48 plant species and assessed their relation with the level of bee dietary specialization. We also investigated whether sterol addition or modification might occur during pollen collection by comparing hand- and bee-collected pollen for nine bee species. Our results show that sterolic composition tends to be similar within the same plant family, but there was no impact of overall relatedness. For pollen sterol content and bee specialization, pollen from plants used by specialist bees displayed more uncommon sterols than pollen from plants used by generalist bees. In addition, a sterol addition behavior may occur during foraging trips and could be considered a possible strategy to balance nutrient deficiencies. Such behavior was unrelated to bee specialization as this phenomenon was observed in both the generalist Apis mellifera and specialist Dasypoda hirtipes, suggesting that sterols might constitute a physiological constraint even for specialist species.