Objectives The role of sex hormones for voice changes in men and women is presently unknown. To determine the effect of sex hormone on the vocal fold, changes of the extracellular matrix (ECM) in vocal fold lamina propria were assessed in orchiectomized (ORX) and ovariectomized (OVX) rats. Methods Male and female Sprague‐Dawley rats were divided into sham‐operated control male (CON‐ORX), ORX, sham‐operated female (CON‐OVX), and OVX rats. Histological changes and expression of ECM‐related genes in lamina propria of the vocal fold were evaluated at 4 and 12 weeks after surgery. Results Testosterone and estradiol levels decreased in the ORX and OVX groups, respectively. ORX groups did not have significant changes compared with CON‐ORX groups. However, the expression of hyaluronic acid (HA) was decreased in the OVX group compared with the CON‐OVX group. The expression of collagen I in OVX was lower than in the CON‐OVX group. Collagen III levels were elevated at 4 weeks in the OVX group, but collagen III levels were diminished at 12 weeks in the OVX group. Expression of elastin in the ECM was less dense in the OVX group compared with controls. The expression MMP‐1 and MMP‐9 showed significantly increase in the OVX group compared to the CON‐OVX group. Conclusion No changes of the ECM‐related genes in the vocal fold lamina propria were observed in ORX groups with reduced testosterone. However, changes of several ECM‐related genes were observed in OVX groups with decreased estrogen. These results indicate that the vocal fold is an estrogen‐sensitive target organ and that decreased estrogen, not testosterone, can affect the expression of several ECM‐related molecules of vocal fold. Level of Evidence NA Laryngoscope, 130:732–740, 2020