The changes in knee laxity and relaxin receptor expression at different phases of rodent estrous cycle are not known. Here, changes in the parameter were investigated in rats at different phases of the estrous cycle. Estrous cycle phases of intact female rats were determined by cytological examination of the vaginal smear. Following phase identification, blood was collected for serum hormone analyses. Knee passive range of motion (ROM) was determined by using a digital miniature goniometer. The animals were then sacrificed and patellar tendon, collateral ligaments and hamstring muscles were harvested for relaxin/insulin-like family peptide receptor 1 and 2 (RXFP1/RXFP2) analyses. Knee passive ROM was the highest at proestrus followed by diestrus and the lowest at estrus. Estrogen level was the highest at proestrus while progesterone and relaxin levels were the highest at diestrus. A strong correlation was observed between relaxin and progesterone levels. At proestrus, expression of RXFP1 and RXFP2 proteins and mRNAs were the highest at proestrus followed by diestrus and estrus. The finding shows that higher level of progesterone and relaxin in diestrus might be responsible for higher laxity of knee joint in rats.