Objective. The aim of this study was to examine the promoting effects of mindfulness training on female college students’ mental toughness in endurance exercise. Methods. A cluster sampling method was used to select 60 female college students as subjects. Based on the body mass index (BMI), stratified randomization was used to divide them into the mindfulness-training group and the control group. Participants in mindfulness-training group had an 8-week mindfulness training, while participants in control group waited. Before and after training, Five Facet Mindfulness Questionnaire (FFMQ) and Connor–Davidson Resilience Scale (CD-RISC) were used for pretest and posttest, and paired t-test and covariance analysis were performed on pretest and posttest between-group data. Results. (1) Paired t-test results showed the posttest scores (26.67 ± 3.56; 20.97 ± 3.66; 126.53 ± 8.59) of the three dimensions of description, nonresponse and FFMQ total score of the mindfulness-training group were higher than the pretest scores (25.53 ± 3.74; 19.23 ± 3.59; 121.43 ± 6.78). Statistical significance was shown in their differences (t = −2.25; −2.70; −3.25,
p
<
0.05
). However, there was no statistical significance in the pretest and posttest of control group. The covariance analyses showed the posttest scores of the mindfulness-training group in three dimensions of description, nonresponse, and FFMQ were higher than the posttest scores of the control group. Statistical significance was shown in their differences (F = 6.55; 6.08; 5.91;
p
<
0.05
). (2) Paired t-test showed posttest scores (46.50 ± 5.93; 30.40 ± 3.75; 15.00 ± 2.34) were significantly higher than pretest scores (42.60 ± 7.68; 26.50 ± 4.32; 12.87 ± 2.51) in all dimensions of the mental toughness of the mindfulness-training group. Statistical significance was shown in their differences (t = −3.135, −4.765, −4.922,
p
<
0.01
). However, there was no significant difference in the pretest and posttest scores in all dimensions of the mental toughness of the control group. The covariance analysis showed that the posttest scores of all dimensions of the mental toughness of the mindfulness-training group were higher than those of the control group, and the differences were statistically significant (F = 11.133, 12.101, 16.053, all
p
<
0.001
). (3) Paired t-test showed that the posttest score of the mindfulness-training group on exercise intensity perception immediately after 800-meter endurance run (5.67 ± 2.61) was lower than the pretest score (7.03 ± 1.24) and the difference was statistically significant (t = 4.18,
p
<
0.001
), while the difference was not statistically significant in the control group. The covariance analysis showed that the posttest score of the mindfulness-training group on exercise intensity perception was lower than that of the control group, and the difference was statistically significant (F = 15.81,
p
<
0.001
). Conclusion. Mindfulness training improved the level of female college students’ mindfulness and mental toughness in their endurance sports, while reducing the fatigue feeling of female college students in endurance sports.