Abstract Toxic tall fescue contains ergot alkaloids, which induce vasoconstriction in livestock, leading to fescue toxicosis. This condition can impact muscle development due to altered blood flow. Supplementation with 5-HTP may counteract the vasoconstrictive effects of fescue alkaloids. We aimed to investigate the influence of 5-HTP supplementation on satellite cells, myoblast, and preadipocyte numbers in maternal and fetal muscle during late gestation in Suffolk ewes. We hypothesized that 5-HTP supplementation would mitigate the effects of fescue toxicosis on muscle cell populations, potentially promoting muscle development and regeneration in maternal and fetal tissues. Suffolk ewes (n = 75), naïve to toxic tall fescue, were blocked by body condition score and body weight into 10 groups for synchronization. Ewes were confirmed pregnant using transabdominal ultrasound, and lamb number was estimated at gestational day (gd) 60. Pregnant ewes carrying twins (n = 30) were randomly assigned to one of three treatments: 1) negative control (CON, n = 10), 2) toxic tall fescue seed containing 1.77 mg ewe-1ּ d-1 ergovaline/ergovalinine plus 5-HTP (2.5 mg/kg BW) supplement (5HTP, n = 10), or 3) positive control, fed toxic tall fescue seed containing 1.77 mg ewe-1ּ d-1 ergovaline/ergovalinine (E+, n = 10). Ewes were individually fed a total mixed ration from gd 78 to gd 85 of pregnancy and then TMR plus treatment (CON, E+ or 5HTP) from gd 86 to d 111. On gd 112, terminal necropsies were performed, and semimembranosus muscle (SM) from the ewe and fetus was removed. Weights were obtained, and then samples were digested using pronase (1.5mg/mL) to isolate preadipocyte (PP1), myoblasts (PP2) and satellite cell (PP3) fractions using a pre-plating technique. Cells were quantified using a Neubauer-hemocytometer and trypan blue to also assess cell viability and changes in cell number. Fetal SM had a greater (P < 0.05) number of pre-adipocytes, myoblasts, and satellite cells per gram of tissue digested in comparison with the ewes. Pre-adipocyte and myoblast number per gram of tissue digested did not differ (P > 0.05) by treatment. The 5HTP group had the greater (P < 0.05) number of satellite cells per gram of tissue digested in comparison with the CON group. Satellite cell numbers tended (P < 0.10) to be greater in the 5HTP group in comparison to the E+ group. Satellite cell numbers from CON and E+ treatment groups did not differ (P > 0.05). In conclusion, 5-HTP supplementation during late gestation in Suffolk ewes exposed to toxic tall fescue significantly increased satellite cell numbers in maternal and fetal muscle tissues. This suggests a potential mitigation of the impact of fescue toxicosis on muscle cell populations.