Lameness in dairy cattle commonly results in milk production losses. Previous studies have quantified this effect in cows milked with conventional milking systems. The effect of lameness on milk production in herds with automatic milking systems (AMS) might differ because lame cows may visit the AMS less frequently, resulting in an additional milk production loss. This observational study aimed to quantify the direct and indirect effect, through milking frequency, of lameness on milk production in AMS herds by means of a multilevel mediation analysis. Lameness scores, the number of visits to the AMS, and daily milk production records of 1608 dairy cows from 87 Dutch AMS herds were available for analysis. Of those cows, 27 % were mildly lame and 11 % were severely lame. Using a multilevel Poisson regression model, both mildly lame and severely lame cows were identified to visit the AMS less frequently. A separate multilevel linear regression model identified that the direct effect of severe lameness on milk production was 1.4 kg per day. The subsequent multilevel mediation analysis showed that the relationship between severe lameness and milk production was mediated through a reduced milking frequency; 63 % of the total milk production losses (3.9 kg per day) were due to a reduced milking frequency (2.5 kg per day). The mediating effect of a reduced milking frequency was most prominent in cows with parity ≥ 4 and in cows that were ≥ 30 days in lactation. This study suggests that milk production losses due to severe lameness of dairy cows might be higher in AMS herds compared with cows in herds with a conventional milking system, a likely result of a reduced milking frequency of those cows. Prevention of severe lameness may be more cost-effective in AMS herds than in herds with a conventional milking system.