In porcine in vitro fertilization (IVF), polyspermy is a persistent obstacle to the efficient production of normal embryos. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) was developed to isolate motile human spermatozoa (from diluted semen by two laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. We previously reported that the monospermy rate was higher when oocytes were co-cultured with isolated spermatozoa in an MFSS for 5 min than when spermatozoa were co-cultured traditionally in drops for 8 h (P < 0.05; Reprod. Fertil. Dev. 20, 187 abst). The present study was undertaken to compare the effect of oocyte location within the MFSS chamber on early development after IVF in the MFSS. A sperm-rich fraction from Berkshire boars was diluted at 1 × 10⁸ cells mL^–1 with modified Modena solution containing 20% seminal fluid. In the first experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine (mm199-caf) for 5 min at room temperature. Before flowing, porcine IVM oocytes were positioned in a part of the MFSS chamber where motile spermatozoa would accumulate with mm199-caf. After flowing for 5 min, those oocytes were cultured in caffeine-free mm199 for 8 h and then in a chemically defined medium (PZM-5) for 7 days. In the second experiment, denuded oocytes were co-cultured with isolated spermatozoa at several locations in the MFSS chamber, and the penetration and monospermy rates were estimated. The concentration of motile spermatozoa was also measured at each place in the MFSS chamber after isolation for 5 min. Statistical analyses of results based on 4 to 5 replicates were carried out by ANOVA and Fisher’s PLSD post hoc test (significance, P < 0.05). When IVM oocytes were co-cultured with spermatozoa gradually accumulated in the chamber of the MFSS for 5 min, the cleavage rate (83.7 ± 6.3% of 121 oocytes) was not different from that of control oocytes co-cultured with spermatozoa (5.7 × 10⁵ cells mL^–1) in 100-μL drops for 5 min (84.6 ± 6.6% of 126 oocytes). However, the blastocyst formation rate (38.2 ± 3.3%) was higher than for the controls (20.6 ± 6.8%; P < 0.05). After flowing for 5 min, the distance from the inflow opening of the MFSS chamber to the location of the oocytes did not affect the sperm penetration rate, but did affect the monospermy rate (14.0 ± 4.0% of 48 oocytes at the nearest position to 50.0 ± 5.6% of 43 oocytes at the furthest position; P < 0.05). After flowing for 5 min, the concentration of motile spermatozoa was also different at each location (57.5 ± 5.6 × 10⁴ cells mL^–1 at the nearest position to 0.8 ± 0.5 × 10⁴ cells mL^–1 at the furthest position; P < 0.05). These observations demonstrate that co-culturing oocytes with spermatozoa that gradually accumulated in the MFSS chamber improved the efficiency of blastocyst formation in the pig, whereas efficiency was affected by the position where oocytes were located in the chamber.