The aims of this study was to evaluate the effect of tonophosphan (TPH), as a phosphorous compound, on resumption of estrus and ovulatory activity of anestrous Egyptian buffalo heifers and anestrous post-partum buffalo cows, in relation with blood phosphorus level. Total of 24 anestrus animals with smooth ovaries and serum progesterone of <1 ng/ml up to 90 days post-partum (12 buffalo cows, weighing 470-530 kg, aging 5-7 years and between 3-4 parities. as well as 12 buffalo heifers, weighing 390-420 kg and 2.5-3 years old) were used in this study. In each of heifer and cow groups (n=12), animals were randomly divided into two sub-groups (treated and control, 6 in each). Animals in treatment group (6 heifers and 6 cows) were injected twice at 7 day-interval with 4 ml/100 kg of TPH, while those in control group (6 heifers and 6 cows) were administrated with 4 ml distilled water/100 kg at the same time of TPH treatment. Estrous activity was detected twice daily to detect estrous signs. Blood samples were collected from all animals of each group on days 0, 3, 6, 10, 14, 18, 22, 26, 30, 34 and 38 of treatment. Results revealed that the estrus rate in treated animals was higher (P<0.05) in cows (83.33%) than in heifers (66.67%), while the control animals showed no estrous signs. Intensity, duration and interval from treatment to estrus were nearly similar in both heifers and cows. Concentration of serum P4 was higher (P<0.05) in cows than in heifers only on days 3 and 6 of treatment, while it was higher (P≥0.05) in cows than in heifers on other sampling days. Concentration of P4 was higher (P<0.05) in treated groups than in control on all sampling days, except during the 1st and six days of treatment. Concentration of P4 was less than 1 ng/ml in control groups on all sampling treatment days, while P4 concentration level was ≥1 ng/ml after 10 days of treatment in treated groups. Concentration of Ca on most sampling days of treatment and P concentration on all sampling days were significantly (P<0.05-P<0.001) higher in heifers than in cows, while Ca:P ratio showed an opposite trend on all sampling days. In conclusion, Based on the foregoing results, twice tonophodphan injection of true anestrous buffalo heifers and cows at a week interval at a level of 4 ml/100 kg LBW has impact on resumption of estrous activity and achieving conception, being more effective for buffalo cows than for buffalo heifers.