Human-animal relationships affect the production and welfare of animals. We investigated whether the type of handling of dairy calves influences their response towards humans. Forty, group-housed, Holstein-Friesian calves were exposed to either positive (e.g. soft voices, slow movements, patting) or negative (e.g. rough voices, rapid movements, pushing) handling (n=20 calves/treatment, 5 calves/group), twice daily (7 min/session) from 4 days to 5 weeks of age. Reactions towards humans were investigated around 4 weeks of age in a 'calf escape test' in their home pens, and by measuring the flight distance in a raceway. In the calf escape test, each calf was given a score between 0 and 4 depending on whether the observer could (1) make eye contact, (2) take 1, or (3) 2 steps towards the calf, or (4) touch the calf before it moved away (defined as moving both forelegs). Data were analysed using ANOVA. Calves that received positive handling showed less avoidance behaviour (mean score; positive:3.7, negative:2.8, sd:0.66, p=0.039) but there was no difference between the treatment groups in flight distance (positive:0.6m, negative:0.7m, sed:0.24m, p=0.526). Calves that received positive handling were 3 times more likely to voluntarily approach a human within 1min, compared to negatively handled calves (50% vs 17% of the calves approached the human, sed: 9.8%, p=0.015). When the calves were 3 months old, we repeated the flight distance and calf escape tests and added a control group (n=20) of the same age that had been reared under normal farm management (minimal handling). Controls showed more avoidance behaviour in the calf escape test (mean score; positive:1.5, negative:1.0, control:0.3, sd:0.21, p<0.001) and greater flight distances (positive:3.3m, negative:3.7m, control:4.9m, sed:0.40m, p<0.001). In conclusion, the type and possibly the amount of handling during early rearing influence the behaviour of calves towards humans, which may have implications for animal welfare.