The study aimed to evaluate in vivo fertility of cryopreserved buffalo spermatozoa by addition of arachidic acid in semen extender. For this purpose, semen was collected from 3 adult buffalo bulls (Bubalus bubalis) of same age by artificial vagina keeping temperature at 42°C for a period of 5 weeks (replicates; n=30). Ejaculates were mixed in tris citric acid extender having 0.0 (control), 20.0, 25.0 and 30.0 ng/mL of arachidic acid at a temperature of 37°C (>1 mL volume, >0.5 billion per mL conc., >60% motility) and cryopreserved using standard procedures. Percent sperm motility, liveability, plasmalemma integrity and viability were increased in extender (P<0.05) having 20.0 ng/mL of arachidic acid compared to 25.0 ng/mL, 30.0 ng/mL and control. However, sperm chromatin integrity was equally improved in experimental extenders having arachidic acid compared to control. Sperm abnormalities were reduced in experimental extender with 20 ng/mL of arachidic acid compared to other experimental extenders containing arachidic acid and control. In experiment 2, a total of 533 inseminations were carried out by the extender containing best level of arachidic acid (20 ng/mL of extender). In vivo fertility was significantly improved in buffaloes inseminated with semen containing 20.0 ng/mL of arachidic acid (58.64%) compared to control (46.06%). In conclusion, addition of arachidic acid (20.0 ng/mL) in extender significantly enhanced quality and in vivo fertility of post thaw buffalo semen.