Gender dysphoria (GD) reflects distress caused by incongruence between one’s experienced gender identity and one’s natal (assigned) gender. Previous studies suggest that high levels of prenatal testosterone (T) in natal females and low levels in natal males might contribute to GD. Here, we investigated if the 2D:4D digit ratio, a biomarker of prenatal T effects, is related to GD. We first report results from a large Iranian sample, comparing 2D:4D in 104 transwomen and 89 transmen against controls of the same natal sex. We found significantly lower (less masculine) 2D:4D in transwomen compared to control men. We then conducted random-effects meta-analyses of relevant studies including our own (k = 6, N = 925 for transwomen and k = 6, N = 757 for transmen). In line with the hypothesized prenatal T effects, transwomen showed significantly feminized 2D:4D (d ≈ 0.24). Conversely, transmen showed masculinized 2D:4D (d ≈ − 0.28); however, large unaccounted heterogeneity across studies emerged, which makes this effect less meaningful. These findings support the idea that high levels of prenatal T in natal females and low levels in natal males play a part in the etiology of GD. As we discuss, this adds to the evidence demonstrating the convergent validity of 2D:4D as a marker of prenatal T effects.