Influenza, a seasonal acute respiratory disease caused primarily by the influenza virus A or B, manifests with severe symptoms leading to considerable morbidity and mortality and is a major concern worldwide. Therefore, effective preventive measures against it are required. The aim of this trial was to evaluate the preventive effects of heat-killed Levilactobacillus brevis KB290 (KB290) in combination with β-carotene (βC) on influenza virus infections in healthy Japanese subjects aged between 20 and 59 y throughout the winter season. We performed a randomized, double-blind, placebo-controlled, parallel-group trial from 16 December 2019 to 8 March 2020, comparing KB290 + βC beverage with placebo beverage. The primary endpoint was the incidence of influenza based on a doctor’s certificate. The incidence of influenza was not significantly different between the two groups. However, the subgroup analysis showed a significant difference between the two groups (influenza incidence: the KB290 + βC group 1.9%, and the placebo group 3.9%) in the subgroup of subjects aged ˂40 y, but not in the subgroup of subjects aged ≥40 y. The results of this trial suggest that the combination of KB290 and βC might be a possible candidate supplement for protection against the seasonal influenza virus infection in humans aged <40 y, although further clinical studies are needed to confirm the concrete preventive effect of this combination on influenza.