Thyroid hormone reportedly induces masculinization of genetic females and goitrogen treatment delays testicular differentiation (ovary-to-testis transformation) in genetic males of Zebrafish. This study explored potential molecular mechanisms of these phenomena. Zebrafish were treated with thyroxine (T4, 2 nM), goitrogen [methimazole (MZ), 0.15 mM], MZ (0.15 mM) and T4 (2 nM) (rescue treatment), or reconstituted water (control) from 3 to 33 days postfertilization (dpf) and maintained in control water until 45 dpf. Whole fish were collected during early (25 dpf) and late (45 dpf) testicular differentiation for transcript abundance analysis of selected male (dmrt1, amh, ar) and female (cyp19a1a, esr1, esr2a, esr2b) sex-related genes by quantitative RT-PCR, and fold-changes relative to control values were determined. Additional fish were sampled at 45 dpf for histological assessment of gonadal sex. The T4 and rescue treatments caused male-biased populations, and T4 alone induced precocious puberty in ∼50% of males. Male-biased sex ratios were accompanied by increased expression of amh and ar and reduced expression of cyp19a1a, esr1, esr2a, and esr2b at 25 and 45 dpf and, unexpectedly, reduced expression of dmrt1 at 45 dpf. Goitrogen exposure increased the proportion of individuals with ovaries (per previous studies interpreted as delay in testicular differentiation of genetic males), and at 25 and 45 dpf reduced the expression of amh and ar and increased the expression of esr1 (only at 25 dpf), esr2a, and esr2b. Notably, cyp19a1a transcript was reduced but via non-thyroidal pathways (not restored by rescue treatment). In conclusion, the masculinizing activity of T4 at the population level may be due to its ability to inhibit female and stimulate male sex-related genes in larvae, while the inability of MZ to induce cyp19a1a, which is necessary for ovarian differentiation, may explain why its “feminizing” activity on gonadal sex is not permanent.