Background Interventions that alter aspects of the physical environments in which unhealthy behaviours occur have the potential to change behaviour at scale, i.e., across populations, and thereby decrease the risk of several diseases. One set of such interventions involves reducing serving sizes, which could reduce alcohol consumption. The effect of modifying the available range of serving sizes of wine in a real-world setting is unknown. We aimed to assess the impact on the volume of wine sold of removing the largest serving size by the glass from the options available in licensed premises. Methods and findings The study was conducted between September 2021 and May 2022 in 21 licensed premises in England that sold wine by the glass in serving sizes greater than 125 ml (i.e., 175 ml or 250 ml) and used an electronic point of sale till system. It used an A-B-A reversal design, set over 3 four-weekly periods. “A” represented the nonintervention periods during which standard serving sizes were served and “B” the intervention period when the largest serving size for a glass of wine was removed from the existing range in each establishment: 250 ml (18 premises) or 175 ml (3 premises). The primary outcome was the daily volume of wine sold, extracted from sales data. Twenty-one premises completed the study, 20 of which did so per protocol and were included in the primary analysis. After adjusting for prespecified covariates, the intervention resulted in −420·8 millilitres (ml) (95% confidence intervals (CIs) −681·4 to −160·2 p = 0·002) or −7·6% (95% CI −12·3%, −2·9%) less wine being sold per day. There was no evidence that sales of beer and cider or total daily revenues changed but the study was not powered to detect differences in these outcomes. The main study limitation is that we were unable to assess the sales of other alcoholic drinks apart from wine, beer, and cider, estimated to comprise approximately 30% of alcoholic drinks sold in participating premises. Conclusions Removing the largest serving size of wine by the glass from those available reduced the volume of wine sold. This promising intervention for decreasing alcohol consumption across populations merits consideration as part of alcohol licensing regulations. Trial registration ISRCTN https://doi.org/10.1186/ISRCTN33169631; OSF https://osf.io/xkgdb.