The core of automatic transplanter is automatically and efficiently picking seedlings. Besides the mechanical structural factors, many other factors, like mechanical properties of seedlings, would affect the successful transplanting rate. Taking cucumber plug seedlings as object, the experiments of clipping and picking seedlings for measuring the picking seedlings force, and gripping seedlings for measuring pressure resistance of seedlings were carried out. The orthogonal experimental design was used to analyze the effect of filling amount, substrate composition, and moisture content on the force of picking seedlings and pressure resistance of seedlings. The experiment results showed that the above three factors all had significant effect on force of picking seedlings and pressure resistance of seedlings. The order from high to low was: filling amount, substrate composition and moisture content for force of picking seedlings, and moisture content, substrate composition and filling amount for pressure resistance of seedlings. The optimum working conditions were as follows: substrate for each cell of 33.5cm3, substrate composition with peat, vermiculitel and perlitel (volume ratio of 3∶1∶1), and moisture content of 65%. These optimum working conditions were accordance with results of comprehensive score obtained by experiment on picking seedling machine.