The preorbital gland plays not only an olfactory role in cervids but also a visual one. Opening this gland is an easy way for the calf to communicate with the mother, indicating hunger/satiety, stress, pain, fear, or excitement. This information can be also useful for farm operators to assess how fast the calves habituate to handling routines and to detect those calves that do not habituate and may suffer chronic stress in the future. Thirty-one calves were subjected to 2 consecutive experiments to clarify if observing preorbital gland opening is related to habituation to handling in red deer calves (Cervus elaphus). Calves were born in 3 different paddocks, handled as newborns (Exp. 1), and then subjected to the same routine handling but with different periodicity: every 1, 2, or 3 wk (Exp. 2). In Exp. 1, preorbital gland opening was recorded in newborns during an initial handling (including weighing, ear tagging, and sex determination). Preorbital gland opening occurred in 93% of calves during this procedure and was not affected by sex, time since birth, or birth weight. Experiment 2 consisted of measuring preorbital opening during the same routine handling (weighing, blood sampling, and rump touching to assess body condition) when calves were 1, 3, and 5 mo old. Binary logistic regression showed that gland opening was associated with habituation to handling, since at 1 and 3 mo the probability of opening the gland decreased with the number of handlings that a calf experienced before (P = 0.008 and P = 0.028, respectively). However, there were no further changes in preorbital gland opening rate in the 5-mo-old calves (P = 0.182). The significant influence of the number of previous handlings on the probability of opening the preorbital gland was confirmed through generalized linear model with repeated measures (P = 0.007). Preorbital gland opening decreased along the phases of the study.