Ectoine has gained considerable attention as a high-value chemical with significant application potential and market demand. This study aimed to increase ectoine yields by blocking the metabolic shunt pathway ofl-aspartate-4-semialdehyde, the precursor substrate in ectoine synthesis. The homoserine dehydrogenase encoded byhominH. campaniensisstrain XH26 is responsible for the metabolic shunt ofl-aspartate-4-semialdehyde to glycine. CRISPR/Cas9 technology was used to seamlessly knockouthom, blocking the metabolic shunt pathway to increase ectoine yields. The ectoine yield of XH26/Δhomwas 351.13 mg (g CDW)−1after 48 h of incubation in 500 mL shake flasks using optimal medium with 1.5 mol L−1NaCl, which was significantly higher than the 239.18 mg (g CDW)−1of the wild-type strain. Additionally, the absence of the ectoine metabolic shunt pathway affects betaine synthesis, and thus the betaine yields of XH26/Δhomwas 19.98 mg (g CDW)−1, considerably lower than the 69.58 mg (g CDW)−1of the wild-type strain. Batch fermentation parameters were optimized, and the wild-type strain and XH26/Δhomwere fermented in 3 L fermenters, resulting in a high ectoine yield of 587.09 mg (g CDW)−1for the defective strain, which was significantly greater than the ectoine yield of 385.03 mg (g CDW)−1of the wild-type strain. This study showed that blocking the metabolic shunt of synthetic substrates effectively increases ectoine production, and a reduction in the competitively compatible solute betaine appears to promote increased ectoine synthesis.