In this study, the effects of heat shock-repair combined with addition of trehalose in lyoprotectant on the freeze-drying resistance and room temperature storage stability of Lactiplantibacillus plantarum LIP-1 were investigated. The results showed that the freeze-drying survival rate of the combined treatment group with heat shock-repair and 4 g/100 mL trehalose addition was 81.65%, which was significantly higher than those of the control group without heat shock-repair treatment (59.92%) and the heat shock-repair treatment group (70.23%) (P < 0.05). The damage degree of cell wall and cell membrane in the combined treatment group was significantly reduced compared with that in the control group. The addition of trehalose maintained a high proportion of unsaturated fatty acids in the cell membrane, reduced cell membrane damage, and significantly enhanced the freeze-drying resistance of the strain while improving its room temperature storage stability. After eight weeks of storage, the survival rate of the combined treatment group was 51.66%, which was significantly higher than those of the control (12.09%) and heat shock-repair (43.79%) groups (P < 0.05), and the intracellular fluorescence intensity was 1969.23 ± 37.22, which was significantly lower than those in the control (3475.21 ± 106.56) and heat shock-repair (2843.95 ± 52.12) groups (P < 0.05). To sum up, the combined treatment could improve the freeze-drying resistance and room temperature storage stability of the strain.