(1) We investigated the effects of the Lactobacillus fermentum HY7302 (HY7302) in a mouse model of benzalkonium chloride (BAC)-induced dry eye, and the possibility of using HY7302 as a food supplement for preventing dry eye. (2) The ocular surface of Balb/c mice was exposed to 0.2% BAC for 14 days to induce dry eye (n= 8), and the control group was treated with the same amount of saline (n= 8). HY7302 (1 × 109CFU/kg/day, 14 days,n= 8) was orally administered daily to the mice, and omega-3 (200 mg/kg/day) was used as a positive control. To understand the mechanisms by which HY7302 inhibits BAC-induced dry eye, we performed an in vitro study using a human conjunctival cell line (clone-1-5c-4). (3) The probiotic HY7302 improved the BAC-induced decreases in the corneal fluorescein score and tear break-up time. In addition, the lactic acid bacteria increased tear production and improved the detached epithelium. Moreover, HY7302 lowered the BAC-induced increases in reactive oxygen species production in a conjunctival cell line and regulated the expression of several apoptosis-related factors, including phosphorylated protein kinase B (AKT), B-cell lymphoma protein 2 (Bcl-2), and activated caspase 3. Also, HY7302 alleviated the expression of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and IL-8, and also regulated the matrix metallopeptidase-9 production in the conjunctival cell line. (4) In this study, we showed thatL. fermentumHY7302 helps prevent dry eye disease by regulating the expression of pro-inflammatory and apoptotic factors, and could be used as a new functional food composition to prevent dry eye disease.