Certain probiotics prevent upper respiratory tract infections (URTIs) by activating immune cells, particularly mononuclear phagocytic cells (MPCs). However, the influence of strain Shirota (LcS), a representative probiotic strain, remains underexplored. This study aimed to investigate the effects of LcS ingestion on URTIs and MPC activation. Two hundred healthy workers aged 23-59 consumed a fermented milk drink containing 4.0 × 10 CFU of LcS (LcS-FM) or a control unfermented milk drink (CM) daily for 28 days during winter. The incidence and severity of URTIs were surveyed using a questionnaire. Peripheral blood mononuclear cells (PBMCs) and serum samples were analyzed for immune cell subsets, cell surface molecules, and cytokines. The LcS-FM group showed a significantly lower incidence and severity of URTIs than the CM group. Notably, the incidence, cumulative incidence, and severity of URTI symptoms were markedly suppressed after 14 days of consumption. LcS-FM also affected MPC activation during this period. The expression of HLA-DR on conventional dendritic cells (cDCs) and monocytes (Mos) was significantly higher in the LcS-FM group on days 14 and 28, along with the expression of CD86 on cDCs on day 14. Among the study participants positive for serum interferon (IFN), the IFNα2 concentration in the LcS-FM group was higher than that in the CM group on day 28. These findings suggest that LcS suppresses the incidence and severity of URTIs in healthy adults, which is associated with the activation of cDCs and Mos.