We previously reported increased release of the excitatory amino acid (EAA) neurotransmitters, glutamate and aspartate, during the early stage of experimental osteoarthritis (OA). Our present objective was to study the effect of intraarticular injection of hyaluronic acid (HA) on OA development, and to analyze concomitant changes in EAA levels in dialysates of anterior cruciate ligament-transected (ACLT) knee joints. OA was induced in Wistar rats by ACLT of one hindlimb; the knee of the other hindlimb was used as the sham-operated control. HA group (n = 12) were injected intraarticularly in the ACLT knee with 1mg of HA once a week for 5 consecutive weeks, starting at 8 weeks after surgery. Saline group (n = 12) were injected as above with normal saline. The sham-operated group, underwent arthrotomy, but not ACLT, and received no treatment (n = 14). Twenty weeks after surgery, knee joint dialysates were collected by microdialysis and EAA levels assayed by high-performance liquid chromatography, and gross morphological examination and histopathological evaluation were performed on the medial femoral condyles and synovia. Rats receiving intraarticular HA injections showed a significantly lower degree of cartilage degeneration on the medial femoral condyle at both the macroscopic level and on the Mankin grading scale than rats receiving saline injections. Intraarticular HA treatment also suppressed synovitis. Moreover, glutamate and aspartate levels were significantly reduced in the HA group compared to the saline group. Intraarticular injection of HA limits articular cartilage and synovium damage and OA formation, and, in parallel, reduces EAA levels in ACLT joint dialysates. This study suggests that the underlying mechanism of the anti-inflammatory effect of HA is to inhibit glutamate and aspartate release in ACLT knee joints, which attenuates the early development of OA.