Purpose: This study was done to examine how laughter therapy impacts serotonin levels, QOL and depression in middle-aged women andto perform a path analysis for verification of the effects. Methods: A quasi-experimental study employing a nonequivalent control group andpre-post design was conducted. Participants were 64 middle-aged women (control=14 and experimental=50 in 3 groups according to levelof depression). The intervention was conducted five times a week for a period of 2 weeks and the data analysis was conducted using repeatedmeasures ANOVA, ANCOVA and LISREL. Results: Results showed that pre serotonin and QOL in women with severe depression werethe lowest. Serotonin in the experimental groups increased after the 10th intervention (p=.006) and the rise was the highest in the group withsevere depression (p=.001). Depression in all groups decreased after the 5th intervention (p=.022) and the biggest decline was observed ingroup with severe depression (p=.007). QOL of the moderate and severe groups increased after the 10th intervention (p=.049), and the increaserate was highest in group with severe depression (pqqqlt;.006). Path analysis revealed that laughter therapy did not directly affect depression,but its effect was indirectly meditated through serotonin variation (pqqqlt;.001). Conclusion: Results indicate that serotonin activationthrough laughter therapy can help middle-aged women by lessening depression and providing important grounds for depression control.