Difructose dianhydride IV (DFA-IV) is produced from levan, which is a natural polysaccharide that belongs to the fructan family, through the activity of levan fructotransferase (LF) derived from microorganisms. Recently, DFA-IV has been expected to have diverse applications in the food and medical industry. Here, we examined the potential application of DFA-IV forin vitro fertilization (IVF) in pigs. In the assessment of acrosomal integrity during incubation, intact acrosomal or viable spermatozoa were highly sustained in 0.1% or 0.25% DFA-IV (69.8%-70.8%,P<0.05). Reactive oxygen species (ROS) levels during sperm incubation decreased following the addition of DFA-IV, and 0.1%-0.5% DFA-IV in particular significantly decreased ROS production relative to that seen with no addition or 0.75% DFA-IV. Total fertilization (mono+ polyspermic oocyte) rate was significantly higher in the addition of 0.1% DFA-IV (94.2%) than with other concentrations (71.8%-86.7%,P<0.05). When using reduced IVF times and lower sperm numbers, we found that addition of 0.1%-0.5% DFA-IV significantly increased the fertilization rate (P<0.05). Fertilized oocytes treated with 0.1% DFA-IV exhibited higher embryonic development and blastocyst formation than those treated with other concentrations (P<0.05). Consequently, the addition of DFA-IV during IVF improved fertilization and embryonic development, suggesting the possible use of novel sugars for enhancement of assisted reproductive technology (ART) in mammals.