The micropylar region of the Rosy barb, Barbus conchonius, egg consists of 7-10 grooves and ridges, which drain directly into a funnel-shaped vestibule, the only point on the chorion through which sperm-egg contact is achieved during fertilization. Results of time-lapse video microscope study and computer-aided analysis of sperm motility pattern in the micropylar region showed that the fertilizing sperm, usually the first to enter the micropylar region, always travelled preferentially along the grooves into the micropylar pit. Subsequently, 86% of sperm arriving the micropylar region within 30 s travelled preferentially along the grooves into the immediate vicinity of the micropylar pit. The sperm guidance role of the micropylar region was calculated to enhance chances of egg penetration/fertilization by as much as 99.7% once sperm were within the micropylar region, possibly in response to some form of chemo-attractant(s) from the egg. Sperm agglutination post-fertilization was also found to occur preferentially along the grooves. Results of our in vitro fertilization experiments showed association between point of sperm entry and blastodisc formation: the blastodisc formed directly beneath the micropyle in all undisturbed eggs.