Serotonin (5‐HT) infusion in vivo causes hypotension and a fall in total peripheral resistance (TPR) that is mediated by 5‐HT7 receptors. 5‐HT dilates small arterioles in skeletal muscle, a microcirculation that importantly contributes to TPR. However, the receptors that mediate this dilation have not been established. Therefore, we tested the hypothesis that 5‐HT7 receptors mediate the arteriolar dilation in the skeletal muscle microcirculation. Cremaster muscles of isoflurane‐anesthetized male Sprague‐Dawley rats were prepared for digital in vivo microscopy and superfused with physiological salt solution (PSS) at 34 °C. Bolus (1‐10 nmols) application of 5‐HT into the superfusate caused substantial dilation of 3rd to 5th order arterioles from 10 ± 1 to 27 ± 1 μm (n = 10 arterioles from 4 rats, p <0.0001) that recovered within 2‐3 min. Dilation to bolus application of 5‐HT was inhibited by superfusion with 1 μM SB269970, a selective 5‐HT7 receptor antagonist (diameter in SB = 9 ± 1 μm; diameter in SB + 5‐HT = 10 ± 1 μm; n = 10 arterioles from 4 rats; p = 0.8763). Similarly, steady‐state superfusion with 10‐30 nM of the 5‐HT1/7 receptor agonist 5‐carboxamidotryptamine (5‐CT) dilated 3rd‐5th‐order arterioles from 16 ± 3 to 37 ± 5 μm (n = 7 arterioles from 3 rats, p = 0.0004). This dilation was reversed (diameter in 1 μM SB269970 = 13 ± 2 μm, n = 7, p = 0.1479 vs. baseline) or blocked (diameter in 1 μM SB269970 + 10‐30 nM 5‐CT = 10 ± 2 μm, n = 7, p = 0.0005 vs. 5‐CT alone) by 1 μM SB269970. Dilation of arterioles to the muscarinic agonist, methacholine (MCH) was not inhibited by SB269970 verifying the specificity of the antagonist; arterioles dilated from 13 ± 2 μm to 25 ± 2 μm to 100 nmols MCH and dilated from 9 ± 2 μm to 32 ± 2 μm in response to MCH in the presence of 1 μM SB269970; n = 10 arterioles from 4 rats; p = 0.7232 vs. dilation in the absence of SB269970. Consistent with these data, pooled samples (4‐5 rats) of 1st and 2nd – order cremaster arterioles expressed message for 5‐HT7 receptors by quantitative real‐time PCR (CT = 36.4 ± 0.74, n = 3 pooled samples, p = 0.039 vs CT = 40). These data support our hypothesis that 5‐HT7 receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to the observed hypotension when these receptors are engaged in vivo.